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Circulating Levels of Corticosterone in the Serum of Developing Chick Embryos and Newly Hatched Chicks
PHYSIOLOGY AND REPRODUCTION Circulating Levels of Corticosterone in the Serum of Developing Chick Embryos and Newly Hatched Chicks T. R. SCOTT, W. A. JOHNSON, D. G. SATTERLEE, and R. P. GILDERSLEEVE Department of Poultry Science, Clyde Ingram Hall, Louisiana State University, Baton Rouge, Louisiana 70803 (Received for publication November 16, 1979)
INTRODUCTION Corticosterone is involved in various roles and interactions in helping maintain homeostasis in the domestic fowl, and its association with metabolism, immunity, and stress response makes this adrenal corticosteroid of interest to the researcher. Furthermore, the study of this steroid and other related corticosteroids in the chick embryo provides information that helps establish the foundation of work done with juvenile and adult chickens. Overall growth and development of the chick embryo, as it is influenced by the administration of adrenal steroid hormones, has been of interest in years past. Administration of cortisone acetate has been shown to inhibit growth and retard development of chick embryos (Karnofsky et al, 1951; Sames and Leathern, 1951; Evans, 1953; Moog and Richardson, 1955; Siegel et al, 1957; and Hall and Kalliecharan, 1975) as has deoxycorticosterone acetate (Sames and Leathern, 1951), Cortisol (Clawson and Domm, 1964), and corticosterone (Wishart et al., 1977). The effects of hypophysectomy, with or without the subsequent introduction of pituitary grafts, have also been used as a means of studying the
involvement of the adrenal glands in ontogenesis of the chick embryo. Fugo (1940) found no apparent effects on the development of the adrenal glands with hypophysectomy of chick embryos, which was the same conclusion of Case (1952) for embryos younger than 16 days of incubation, but not for those older than this age. A reduction in adrenal weight was found by Betz (1967) at 20 days of incubation in both hypophysectomized embryos and hypophysectomized embryos with pituitary grafts; Girouard and Hall (1973) noted reduced adrenal weights and cortical mitotic activity as a result of hypophysectomy or dexamethasone administration after 11 days of incubation. The latter two studies concluded there was an embryonic relationship between the developing pituitary and adrenal glands. Hall (1970, 1971) examined 14-day-old and older embryos with grafts of additional adrenal glands to have less cortical mitotic activity, which was attributed to a negative feedback on ACTH release from the host pituitary gland. Pedernera (1971, 1972) was able to discern that embryonic adrenal glands cultured in vitro become responsive to ACTH at as early as 5 days of incubation.
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The presence of increasing levels of cortico-
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ABSTRACT Circulating levels of corticosterone were determined in chick embryos from 10 to 21 days of incubation using eggs from a Leghorn breeder flock. In Experiment 1, eggs were incubated from 10 to 20 days for daily embryonic blood collection. To verify stage of development with day of incubation, embryo right middle toe lengths were measured concurrent with blood sampling. Serum from three embryos was pooled into one sample and the corticosterone content of 10 samples per day of incubation was determined using a radioimmunoassay procedure. The levels of corticosterone from day 10 to 14 fluctuated slightly and then increased rapidly until 16 days of incubation. At this time serum corticosterone remained relatively constant through day 18 with an apparent increasing trend up to day 20. The use of toe lengths to assure no day-to-day overlap in embryonic development proved effective. In Experiment 2, newly hatched (day 21) chicks were sorted into four 3-hr periods ranging from early to late hatching. Blood samples were collected from five individual chicks during four 15-min sampling periods for each of the four hatch times. Serum corticosterone levels were not affected by sampling periods or hatch times. (Key words: corticosterone, embryos, radioimmunoassay, hatching time) 1981 Poultry Science 60:1314-1320
CORTICOSTERONE IN CHICK EMBRYOS
In the time after chicks hatch, researchers have measured plasma corticosterone levels on day 1 (Wise and Frye, 1973; Gildersleeve et al, 1978; Nakamura et al, 1978; Satterlee et al, 1980) and 2 days posthatch (Kalliecharan and Hall, 1974; Siegel and Gould, 1976). Overall, plasma corticosterone concentrations decline considerably after a peak that occurs at the onset and during hatching. Gildersleeve et al (1978) and Satterlee et al. (1980) have shown that newly hatched chicks exhibit day 1 fluctuations of plasma corticosterone. Therefore, depending on the time of day blood of neonate chicks is sampled, it can be supposed that corticosterone levels will be found to either increase or decrease after hatching. The present investigation reports the use of a radioimmunoassay procedure to determine the serum levels of corticosterone circulating in developing chick embryos sequentially from 10 days of incubation through hatching. Corticosterone in the serum of hatched chicks was determined to study the effect of time of blood sampling and hatching on hormone level. MATERIALS AND METHODS Two groups of fertile eggs were obtained from a Leghorn breeder flock and incubated in a Petersime Model 4 incubator at 37 C and 56% relative humidity. One group of eggs was incubated from 10 to 20 days for embryonic blood
sampling; the other group of eggs was allowed to incubate to term for blood collection from hatched chicks. Blood sampling (Experiment 1) was carried out on 30 individual embryos on subsequent days from day 10 to 20 of incubation at approximately 0900 hr. Embryos were removed from eggs and blood was collected directly into 10 x 75 mm culture tubes from the vitelline artery -and vein. Blood sampling of all embryos was completed within a 20 min period. This time period was in accordance with the stress response study done by Wise and Frye (1973) which showed, at 20 min after opening the shell and breaking the shank of one leg, embryos with intact adenohypophyseal-adrenal axes responded significantly to this stressor. Individual blood samples were allowed to stand at room temperature until clotting occurred. The samples were then centrifuged at 886 g for 20 min. After centrifugation, serum samples were pooled into 10 groups of three embryos per group and stored frozen until assayed for corticosterone using a radioimmunoassay procedure. After blood sampling, individual embryo right middle toe lengths were measured (Hamburger and Hamilton, 1951) as a means of verifying age within a sampling period so as to eliminate any possible day-to-day overlap in embryonic development. Previous work (Gildersleeve et al., 1978; Satterlee et al., 1980) in our laboratory had involved measuring plasma corticosterone levels of neonate chicks over a 24 hr period. Since these chicks had been obtained from a commercial hatchery, there was no way of knowing if the group as a whole had hatched together in the same hatchery unit or if they represented neonates ranging in ages anywhere up to 12 hr posthatching. With this in mind, it was decided that Experiment 2 should examine the possibility of a posthatching age effect on circulating corticosterone levels. In Experiment 2, the second group of eggs was allowed to incubate normally until day 20. At this time, the detection of pipped shells was used as a criterion for separation of the eggs into four hatching groups which were termed very early, early, late, and very late hatching chicks. This separation procedure allowed for the sampling of neonates that ranged up to 12 hr posthatch in age. On day 2 1 , 5 neonate chicks from each of the four hatching groups were blood sampled by decapitation during each of four 15-min periods within an hour.
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steroids in amnionic fluid (Woods et al, 1971) and plasma (Kalliecharan and Hall, 1974) occurring with developmental age have been measured with a fluorometric technique and competitive protein binding assay procedures, respectively. The competitive protein binding assay has also been used to measure the progressively increasing plasma corticosterone levels in developing chick embryos up to and through hatching (Wise and Frye, 1973; Siegel and Gould, 1976). Nakamura et al. (1978) have indicated that they have used a radioimmunoassay procedure to measure plasma corticosterone in 17 and 21-day-old embryos, and 3, 7, and 14-day-old chicks. Some of these researchers (Woods et al, 1971; Wise and Frye, 1973; Kalliecharan and Hall, 1974) also have proposed the time during chick embryonic development when the adenohypophysealadrenal axis is established. Depending on which reference is consulted, this period of time ranges between the 14th and 16th day of incubation.
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Daily serum corticosterone levels and right middle toe lengths of developing chick embryos (Experiment 1) were analyzed by analysis of
TABLE 1. Serum corticosterone concentrations and right middle toe lengths by day of incubation for chick embryos and neonates Serum corticosterone1i,b
Toe lengths a
(ng/ml)
(cm)
3.61 + .51 c 2.99 ± .42 3.45 + .71 3.22 ± .37 3.94 ± .45 5.80 ± .42 6.70 ± .72 6.64 ± .87 6.41 + :1.11 7.40 ± .75 9.27+ :1.16 9.04 ± .42
.41 .58 .74 .86 1.02 1.17 1.38 1.61 1.78 1.86 2.02
± ± ± ± ± ± ± + ± ± ±
.01 c .01 .01 .01 .01 .02 .03 .02 .02 .03 .03
Number of observations for days 10 to 20 = 10. Number of observations for day 21 = 79. c
Mean ± SEM.
variance using the following model: Y ;j = u + D ; + e ;j where, Yy = serum corticosterone or toe length the jth pooled embryonic sample on ith day u = overall mean Dj = ith day, i = 10 to 16 or 15 to 20 corticosterone levels and 10 to 20 for lengths ejj = random error
for the
for toe
Corticosterone levels were additionally subjected to polynomial regression and mean toe lengths between consecutive days were further tested with preplanned LSD comparisons. The corticosterone levels of hatched chicks (day 21) were analyzed by analysis of variance using the following model: Y i j k = u + Tj + Pj + T;Pj + e i j k where, Yjj k = serum corticosterone for the ith hatching time, jth sampling period, and kth neonate u = overall mean Tj = ith hatching time, i = 1 to 4 P: = jth sampling period, j = 1 to 4 TjPj = ith hatching time, jth sampling period interaction e - k = random error RESULTS AND DISCUSSION
Serum corticosterone levels of chick embryos from day 10 of incubation through hatching are shown in Table 1 and are graphically represented in Figure 1. The confidence in stating that a particular mean level of corticosterone corresponds to a certain day of incubation requires staging embryos for development. In an effort to reduce variation among individual embryos, especially during the later phase of incubation prior to hatching, the staging method of Hamburger and Hamilton (1951) was followed. Mean right middle toe lengths are given in Table 1. The LSD comparisons revealed that mean toe lengths from day-to-day were significantly different (P<.01), and this was expected
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The handling of blood samples in preparation for the corticosterone radioimmunoassay procedure was the same as that discussed for Experiment 1. The radioimmunoassay procedure used was a modification of the Endocrine Sciences radioimmunoassay for corticosterone (Endocrine Sciences, 1972) and has been previously described (Satterlee et al., 1980). Serum samples were consecutively extracted with 2,2,4-trimethylpentane and dichloromethane. Corticosterone was assayed in the latter extract. The extraction efficiency for corticosterone from pooled samples of embryonic and hatched chick serum was found to be 88.17 + .37% (n = 48). This extraction efficiency was determined on pooled serum samples from all embryonic and hatch chick groups, and there was no difference in efficiencies among days of incubation or the day of hatching. Intrassay variation for three assays of embryonic serum samples were 2.34%, 5.16%, and 5.37%; and interassay variation across all three assays was determined to be 23.79%. The assay of neonate serum samples had an intrassay variation of 10.61%.
CORTICOSTERONE IN CHICK EMBRYOS n
10
II
E
13
14
15
16
17
18
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20
21
DAYS OF INCUBATION
FIG. 1. Serum concentrations (means ± SEM) of corticosterone from developing chick embryos and neonates. Solid lines are representative of the regression relationships fitted to the data.
since the intent of staging the embryos was to select them effectively on the basis of similarity of development. The levels of corticosterone were examined within each half of the incubation period covered in this study since previous work (Woods et al, 1971; Wise and Frye, 1973; Kalliecharan and Hall, 1974, 1976) indicates that the adrenal glands are autonomous in their secretion of corticosterone prior to about 15 to 16 days of incubation and under the control of the adenohypophysis thereafter. Serum corticosterone levels for days 10 through 16 of incubation, constituting the first half of the period studied, were different (P< .01), and a trend analysis revealed that a quadratic relationship existed for the levels of corticosterone. From the data, a quadratic function was calculated for the mean corticosterone of days 10 through 16 with the following equation:
discussed by Woods et al. (1971), Wise and Frye (1973), and Kalliecharan and Hall (1974, 1976). The pattern of corticosterone secretion reported in this study for the period of time from 10 through 16 days of incubation is similar to those reported by Wise and Frye (1973), Kalliecharan and Hall (1974), and Siegel and Gould (1976), who all measured plasma corticosterone with a competitive protein binding assay. During the time from 10 to 16 days of incubation, Wise and Frye (1973) reported plasma corticosterone values that were approximately one-fourth of those reported here. Kalliecharan and Hall (1974) reported levels approximately tenfold higher, and Siegel and Gould (1976) also graphically showed plasma corticosterone concentrations to be higher than the values of this study. Girouard and Hall (1973) reported that the mitotic activity of the adrenal gland is greatest on day 13 of incubation, and Hall (1970) stated that mitotic activity in the embryonic adrenal gland peaked on day 14. In light of these observations, the increasing presence of corticosterone in the serum of the embryos sampled from 14 to 16 days may be reflective of the increased number of secretory cells in the adrenal cortex at 13 or 14 days of incubation. The increasing secretory activity of the embryonic adrenal gland has been documented by Kalliecharan and Hall (1976), and the peak concentration of total corticosteroids in adrenal homogenates at 15 days of incubation was noted to correspond to the rapid synthesis and secretion of steroids through possible stimulation of the adrenals by the pituitary gland. Statistical analysis revealed no differences among the corticosterone values from 15 through 20 days of incubation (Table 1), and a linear relationship was fitted to the data for graphic presentation (Fig. 1). This linear relationship was calculated from the following equation:
q = 29.486 - 4.55D ; + .196D? q = - 2 . 5 7 2 + .549D; where C; = corticosterone level for the ith day, Dj = ith day, i = 10 to 16. This is graphically depicted in Figure 1, which shows that the levels fluctuate slightly from 10 to 14 days and then increase until day 16. In and around the 15th or 16th day of incubation, the secretion of corticosterone is possibly brought under control by the establishment of the adenohypophyseal-adrenal axis, which has been
where C; and Dj correspond to the designations given them for the previous equation and i = 15 to 20. Values reported here for the period of time from 15 through 20 days of incubation averaged 7.04 ng/ml. Wise and Frye (1973) found corticosterone levels one-third lower, but Kalliecharan and Hall (1974) reported values about threefold higher in embryonic
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Wise and Frye (1973) believed that increasing plasma corticosterone levels prior to hatching were the result of a hyperactive adrenal or lowered turnover rate. These researchers and Siegel and Gould (1976) indicated that it is necessary for plasma corticosterone levels to increase prior to the onset of hatching for duodenal epithelium differentiation, and for increases in alkaline phosphatase levels which were studied by Moog and Richardson (1955). Kalliecharan and Hall (1974) believed that a decrease in corticosteroid concentrations in the embryonic plasma prior to hatching is representative of increased hormone metabolism, decreased synthesis, and decreased secretion at that time. Corticosterone binding capacity (Siegel and Gould, 1976; Martin et al., 1977; Gasc and Martin, 1978) has been shown to decrease after 15 or 16 days of incubation allowing for more circulating free or active corticosterone perhaps necessary for the onset of hatching. However, if the amount of corticosterone (or glucocorticoids) present is too great, delayed hatching time, decreased hatchability,
and improper yolk sac retraction will occur (Brooks and Ungar, 1967; Wishart et al, 1977). When the chick neonate hatches, the presence of large concentrations of circulating corticosterone and other glucocorticoids are suspected since this initial emergence is believed to be quite eventful for the chick. A graphic representation of the mean levels of corticosterone determined from a group of chicks ranging up to 12 hr posthatch in age is shown in Figure 2. In order to show the degree of variation (possibly due to episodic release) associated with sampling chicks within an hourly interval, the four 15-min sampling periods were included. Analysis of these data indicated no differences existed among any of the hatching groups or the sampling periods. This indicates that within a 12 hr posthatch time range, blood sampling for corticosterone determination can be done on a group of neonate chicks without detection of differences in corticosterone levels if hatching time has been disregarded. Gildersleeve et al. (1978) and Satterlee et al. (1980) used the radioimmunoassay procedure employed in the present study to measure the first 24 hr fluctuations of plasma corticosterone in commercially obtained broiler chicks which had been debeaked and vaccinated at the
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P2 P3 P4 PI P2 P3 P4 PI P2 P3 P4 PI P2 P3 P4 HI H2 H3 H4
HATCHING TIMES (H) AND SAMPLING PERIODS (P)
FIG. 2. Serum concentrations (mean ± SEM) of corticosterone from neonate chicks. Hatching times are as follows: HI = 10 to 12 hr posthatch, H2 = 7 to 9 hr posthatch, H3 = 4 to 6 hr posthatch, H4 = 1 to 3 hr posthatch. Sampling periods are as follows: PI = first 15 min of hour, P2 = second IS min of hour, P3 = third 15 min of hour, P4 = fourth 15 min of hour.
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plasma. Siegel and Gould (1976) also found higher levels than those presented here, and the only other measure of corticosterone with a radioimmunoassay (Nakamura et al., 1978) found 17-day-old embryos to have levels of 7.20 ng/ml. No differences among serum corticosterone concentrations from 15 through 20 days of incubation may be indicative of the increasing divergence among embryos in preparation for hatching, or perhaps there is no reason for serum corticosterone levels to increase significantly prior to hatching. Although the day 15 through 20 corticosterone values did not vary statistically, an upwards trend to day 20 was apparent. Indeed, the serum corticosterone value at day 20 was elevated 39% over the day 15 value. A comparison of the standard errors associated with the corticosterone means in both periods of the incubation time studied here reveals that more variation is found during that time when the adenohypophysis is believed to be influencing corticosterone secretion. The assumption that the adrenal gland is autonomous in its secretion of corticosterone prior to day 15 or 16 has already been made; and the continuance of this autonomy with superimposed adenohypophyseal influences could perhaps explain the larger degree of variation observed during the time just before and through day 20.
CORTICOSTERONE IN CHICK EMBRYOS
ACKNOWLEDGMENTS T h e senior a u t h o r thanks A. B. Watts, J. A. Hebert, Jr., a n d E. W. Byrd, Jr., for their instruction, advice, a n d review of this m a n u script. T h e help of Stephanie G. Scott, W. T. Springer, R. J. Daigle, and Marlene Ochoa is very m u c h appreciated, and t h e a u t h o r also t h a n k s Linda J o e for t y p i n g t h e m a n u s c r i p t .
REFERENCES Betz, T. W., 1967. The effects of embryonic pars distalis grafts on the development of hypophysectomized chick embryos. Gen. Comp. Endocrinol. 9:172-186. Brooks, W. S., and F. Ungar, 1967. Effects of C „ methyl steroids on the musculus complexus and hatching of the chick. Proc. Soc. Exp. Biol. Med. 125:488-492. Case, J. F., 1952. Adrenal cortical-anterior pituitary relationships during embryonic life. Ann. NY Acad. Sci. 55:147-158. Clawson, R. C , and L. V. Domm, 1964. Growth and liver glycogen in the chick embryo under the influence of corticosteroids. Physiol. Zool. 37: 307-315. Endocrine Sciences, 1972. Plasma corticosterone radioimmunoassay procedure, antiserum B 2142. 18418 Oxnard Street, Tarzana, CA. Evans, H. J., 1953. Action of cortisone on developing chick embryo. Proc. Soc. Exp. Biol. Med. 83: 31-34. Fugo, N. W., 1940. Effects of hypophysectomy in the chick embryo. J. Exp. Zool. 85:271-297. Gasc, J. M., and B. Martin, 1978. Plasma corticosterone binding capacity in the partially decapitated chick embryo. Gen. Comp. Endocrinol. 35: 274-279. Gildersleeve, R. P., D. G. Satterlee, and W. A. Johnson, 1978. Plasma glucocorticoid levels in broiler chicks using two blood sampling techniques. Poultry Sci. 57:1138. Girouard, R. J., and B. K. Hall, 1973. Pituitary-adrenal
interactions and growth of the embryonic avian adrenal gland. J. Exp. Zool. 183:323-332. Hall, B. K., 1970. Response of host embryonic chicks to grafts of additional adrenal glands. 1. Response of host adrenal glands, gonads, and kidneys. Can. J. Zool. 48:867-872. Hall, B. K., 1971. Response of host embryonic chicks to grafts of additional adrenal glands. 3. Response of adrenal glands and gonads to the pressence of homogenates of adrenal glands. Can. J. Zool. 49:381-384. Hall, B. K., and R. Kalliecharan, 1975. The effects of exogenous cortisone acetate on development, especially skeletal development, and on circulating levels of corticosteroids in chick embryos. Teratology 12:111-119. Hamburger, V., and H. L. Hamilton, 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 8 8 : 4 9 - 9 2 . Kalliecharan, R., and B. K. Hall, 1974. A developmental study of the levels of progesterone, corticosterone, Cortisol, and cortisone circulating in plasma of chick embryos. Gen. Comp. Endocrinol. 24:364-372. Kalliecharan, R., and B. K. Hall, 1976. A developmental study of progesterone, corticosterone, Cortisol, and cortisone in the adrenal gland of the embryonic chick. Gen. Comp. Endocrinol. 30: 404-409. Karnofsky, D. A., L. P. Ridgeway, and P. A. Patterson, 1951. Growth inhibiting effect of cortisone acetate on the chick embryo. Endocrinology 48: 596-616. Martin, B., J. M. Gasc, and M. Thibier, 1977. C 21 steroid binding proteins and progesterone levels in chicken plasma during ontogenesis. J. Steroid Biochem. 8:161-166. Moog, F., and D. Richardson, 1955. The influence of adrenocortical hormones on differentiation and phosphate synthesis in the duodenum of the chick embryo. J. Exp. Zool. 130:29—56. Nakamura, T., Y. Tanabe, and H. Hirano, 1978. Evidence of the in vitro formation of Cortisol by the adrenal gland of embryonic and young chickens (Gallus domesticus). Gen. Comp. Endocrinol. 35:302-308. Pedernera, E. A., 1971. Development of the secretory capacity of the chick embryo adrenal glands. J. Embryol. Exp. Morphol. 25:213-222. Pedernera, E. A., 1972. Adrenocorticotropic activity in vitro of the chick embryo pituitary gland. Gen. Comp. Endocrinol. 19:589-590. Sames, H. L., and J. H. Leathern, 1951. Influence of desoxycorticosterone acetate and cortisone acetate on body weight of chick embryos. Proc. Soc. Exp. Biol. Med. 78:231-232. Satterlee, D. G., R. B. Abdullah, and R. P. Gildersleeve, 1980. Plasma corticosterone radioimmunoassay and levels in the neonate chick. Poultry Sci. 59:900-905. Siegel, B. V., M. J. Smith, and B. Gersd, 1957. Effects of cortisone on the developing chick embryo. Arch. Pathol. 63:562-570. Siegel, H. S., and N. R. Gould, 1976. Chick embryonic plasma proteins and binding capacity for corticosterone. Dev. Biol. 50:510-516. Wise, P. M., and B. E. Frye, 1973. Functional develop-
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h a t c h e r y . These researchers f o u n d highest levels at the h a t c h e r y (some 2 0 ng/ml) a n d 2 0 h r later (some 11 ng/ml) w i t h lowest levels (some 6 n g / m l ) at 1 0 t o 1 4 h r after receiving t h e chicks. In light of these results, t h e mean level presented for day 21 in this s t u d y (Table 1, Fig. 1) can only be assumed t o represent t h e level of corticosterone in t h e serum at t h e particular t i m e of day w h e n t h e samples were taken from t h e n e o n a t e s . If a m e a n value can be used t o represent t h e level of corticosterone of chicks hatching a t various t i m e s up t o 12 hr after first emergence, t h e n it m a y be supposed t h a t t h e activity of those chicks already h a t c h e d influences t h e levels of corticosterone of chicks j u s t h a t c h e d and hatching.
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ment of the hypothalamo-hypophyseal-adrenal cortex axis in the chick embryo, Gallus domesticus. J. Exp. Zool. 185:277-292. Wishart, G. J., J. E. A. Leakey, and G. J. Dutton, 1977. Differential effects of hormones on precocious yolk sac retraction in chick embryos fol-
lowing administration by a new technique. Gen. Comp. Endocrinol. 31:373-380. Woods, J. E., G. W. DeVries, and R. C. Thommes, 1971. Ontogenesis of the pituitary-adrenal axis in the chick embryo. Gen. Comp. Endocrinol. 17: 407-415.
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INTRODUCTION Corticosterone is involved in various roles and interactions in helping maintain homeostasis in the domestic fowl, and its association with metabolism, immunity, and stress response makes this adrenal corticosteroid of interest to the researcher. Furthermore, the study of this steroid and other related corticosteroids in the chick embryo provides information that helps establish the foundation of work done with juvenile and adult chickens. Overall growth and development of the chick embryo, as it is influenced by the administration of adrenal steroid hormones, has been of interest in years past. Administration of cortisone acetate has been shown to inhibit growth and retard development of chick embryos (Karnofsky et al, 1951; Sames and Leathern, 1951; Evans, 1953; Moog and Richardson, 1955; Siegel et al, 1957; and Hall and Kalliecharan, 1975) as has deoxycorticosterone acetate (Sames and Leathern, 1951), Cortisol (Clawson and Domm, 1964), and corticosterone (Wishart et al., 1977). The effects of hypophysectomy, with or without the subsequent introduction of pituitary grafts, have also been used as a means of studying the
involvement of the adrenal glands in ontogenesis of the chick embryo. Fugo (1940) found no apparent effects on the development of the adrenal glands with hypophysectomy of chick embryos, which was the same conclusion of Case (1952) for embryos younger than 16 days of incubation, but not for those older than this age. A reduction in adrenal weight was found by Betz (1967) at 20 days of incubation in both hypophysectomized embryos and hypophysectomized embryos with pituitary grafts; Girouard and Hall (1973) noted reduced adrenal weights and cortical mitotic activity as a result of hypophysectomy or dexamethasone administration after 11 days of incubation. The latter two studies concluded there was an embryonic relationship between the developing pituitary and adrenal glands. Hall (1970, 1971) examined 14-day-old and older embryos with grafts of additional adrenal glands to have less cortical mitotic activity, which was attributed to a negative feedback on ACTH release from the host pituitary gland. Pedernera (1971, 1972) was able to discern that embryonic adrenal glands cultured in vitro become responsive to ACTH at as early as 5 days of incubation.
1314
The presence of increasing levels of cortico-
Downloaded from http://ps.oxfordjournals.org/ at Emory University Libraries on April 18, 2015
ABSTRACT Circulating levels of corticosterone were determined in chick embryos from 10 to 21 days of incubation using eggs from a Leghorn breeder flock. In Experiment 1, eggs were incubated from 10 to 20 days for daily embryonic blood collection. To verify stage of development with day of incubation, embryo right middle toe lengths were measured concurrent with blood sampling. Serum from three embryos was pooled into one sample and the corticosterone content of 10 samples per day of incubation was determined using a radioimmunoassay procedure. The levels of corticosterone from day 10 to 14 fluctuated slightly and then increased rapidly until 16 days of incubation. At this time serum corticosterone remained relatively constant through day 18 with an apparent increasing trend up to day 20. The use of toe lengths to assure no day-to-day overlap in embryonic development proved effective. In Experiment 2, newly hatched (day 21) chicks were sorted into four 3-hr periods ranging from early to late hatching. Blood samples were collected from five individual chicks during four 15-min sampling periods for each of the four hatch times. Serum corticosterone levels were not affected by sampling periods or hatch times. (Key words: corticosterone, embryos, radioimmunoassay, hatching time) 1981 Poultry Science 60:1314-1320
CORTICOSTERONE IN CHICK EMBRYOS
In the time after chicks hatch, researchers have measured plasma corticosterone levels on day 1 (Wise and Frye, 1973; Gildersleeve et al, 1978; Nakamura et al, 1978; Satterlee et al, 1980) and 2 days posthatch (Kalliecharan and Hall, 1974; Siegel and Gould, 1976). Overall, plasma corticosterone concentrations decline considerably after a peak that occurs at the onset and during hatching. Gildersleeve et al (1978) and Satterlee et al. (1980) have shown that newly hatched chicks exhibit day 1 fluctuations of plasma corticosterone. Therefore, depending on the time of day blood of neonate chicks is sampled, it can be supposed that corticosterone levels will be found to either increase or decrease after hatching. The present investigation reports the use of a radioimmunoassay procedure to determine the serum levels of corticosterone circulating in developing chick embryos sequentially from 10 days of incubation through hatching. Corticosterone in the serum of hatched chicks was determined to study the effect of time of blood sampling and hatching on hormone level. MATERIALS AND METHODS Two groups of fertile eggs were obtained from a Leghorn breeder flock and incubated in a Petersime Model 4 incubator at 37 C and 56% relative humidity. One group of eggs was incubated from 10 to 20 days for embryonic blood
sampling; the other group of eggs was allowed to incubate to term for blood collection from hatched chicks. Blood sampling (Experiment 1) was carried out on 30 individual embryos on subsequent days from day 10 to 20 of incubation at approximately 0900 hr. Embryos were removed from eggs and blood was collected directly into 10 x 75 mm culture tubes from the vitelline artery -and vein. Blood sampling of all embryos was completed within a 20 min period. This time period was in accordance with the stress response study done by Wise and Frye (1973) which showed, at 20 min after opening the shell and breaking the shank of one leg, embryos with intact adenohypophyseal-adrenal axes responded significantly to this stressor. Individual blood samples were allowed to stand at room temperature until clotting occurred. The samples were then centrifuged at 886 g for 20 min. After centrifugation, serum samples were pooled into 10 groups of three embryos per group and stored frozen until assayed for corticosterone using a radioimmunoassay procedure. After blood sampling, individual embryo right middle toe lengths were measured (Hamburger and Hamilton, 1951) as a means of verifying age within a sampling period so as to eliminate any possible day-to-day overlap in embryonic development. Previous work (Gildersleeve et al., 1978; Satterlee et al., 1980) in our laboratory had involved measuring plasma corticosterone levels of neonate chicks over a 24 hr period. Since these chicks had been obtained from a commercial hatchery, there was no way of knowing if the group as a whole had hatched together in the same hatchery unit or if they represented neonates ranging in ages anywhere up to 12 hr posthatching. With this in mind, it was decided that Experiment 2 should examine the possibility of a posthatching age effect on circulating corticosterone levels. In Experiment 2, the second group of eggs was allowed to incubate normally until day 20. At this time, the detection of pipped shells was used as a criterion for separation of the eggs into four hatching groups which were termed very early, early, late, and very late hatching chicks. This separation procedure allowed for the sampling of neonates that ranged up to 12 hr posthatch in age. On day 2 1 , 5 neonate chicks from each of the four hatching groups were blood sampled by decapitation during each of four 15-min periods within an hour.
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steroids in amnionic fluid (Woods et al, 1971) and plasma (Kalliecharan and Hall, 1974) occurring with developmental age have been measured with a fluorometric technique and competitive protein binding assay procedures, respectively. The competitive protein binding assay has also been used to measure the progressively increasing plasma corticosterone levels in developing chick embryos up to and through hatching (Wise and Frye, 1973; Siegel and Gould, 1976). Nakamura et al. (1978) have indicated that they have used a radioimmunoassay procedure to measure plasma corticosterone in 17 and 21-day-old embryos, and 3, 7, and 14-day-old chicks. Some of these researchers (Woods et al, 1971; Wise and Frye, 1973; Kalliecharan and Hall, 1974) also have proposed the time during chick embryonic development when the adenohypophysealadrenal axis is established. Depending on which reference is consulted, this period of time ranges between the 14th and 16th day of incubation.
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Daily serum corticosterone levels and right middle toe lengths of developing chick embryos (Experiment 1) were analyzed by analysis of
TABLE 1. Serum corticosterone concentrations and right middle toe lengths by day of incubation for chick embryos and neonates Serum corticosterone1i,b
Toe lengths a
(ng/ml)
(cm)
3.61 + .51 c 2.99 ± .42 3.45 + .71 3.22 ± .37 3.94 ± .45 5.80 ± .42 6.70 ± .72 6.64 ± .87 6.41 + :1.11 7.40 ± .75 9.27+ :1.16 9.04 ± .42
.41 .58 .74 .86 1.02 1.17 1.38 1.61 1.78 1.86 2.02
± ± ± ± ± ± ± + ± ± ±
.01 c .01 .01 .01 .01 .02 .03 .02 .02 .03 .03
Number of observations for days 10 to 20 = 10. Number of observations for day 21 = 79. c
Mean ± SEM.
variance using the following model: Y ;j = u + D ; + e ;j where, Yy = serum corticosterone or toe length the jth pooled embryonic sample on ith day u = overall mean Dj = ith day, i = 10 to 16 or 15 to 20 corticosterone levels and 10 to 20 for lengths ejj = random error
for the
for toe
Corticosterone levels were additionally subjected to polynomial regression and mean toe lengths between consecutive days were further tested with preplanned LSD comparisons. The corticosterone levels of hatched chicks (day 21) were analyzed by analysis of variance using the following model: Y i j k = u + Tj + Pj + T;Pj + e i j k where, Yjj k = serum corticosterone for the ith hatching time, jth sampling period, and kth neonate u = overall mean Tj = ith hatching time, i = 1 to 4 P: = jth sampling period, j = 1 to 4 TjPj = ith hatching time, jth sampling period interaction e - k = random error RESULTS AND DISCUSSION
Serum corticosterone levels of chick embryos from day 10 of incubation through hatching are shown in Table 1 and are graphically represented in Figure 1. The confidence in stating that a particular mean level of corticosterone corresponds to a certain day of incubation requires staging embryos for development. In an effort to reduce variation among individual embryos, especially during the later phase of incubation prior to hatching, the staging method of Hamburger and Hamilton (1951) was followed. Mean right middle toe lengths are given in Table 1. The LSD comparisons revealed that mean toe lengths from day-to-day were significantly different (P<.01), and this was expected
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The handling of blood samples in preparation for the corticosterone radioimmunoassay procedure was the same as that discussed for Experiment 1. The radioimmunoassay procedure used was a modification of the Endocrine Sciences radioimmunoassay for corticosterone (Endocrine Sciences, 1972) and has been previously described (Satterlee et al., 1980). Serum samples were consecutively extracted with 2,2,4-trimethylpentane and dichloromethane. Corticosterone was assayed in the latter extract. The extraction efficiency for corticosterone from pooled samples of embryonic and hatched chick serum was found to be 88.17 + .37% (n = 48). This extraction efficiency was determined on pooled serum samples from all embryonic and hatch chick groups, and there was no difference in efficiencies among days of incubation or the day of hatching. Intrassay variation for three assays of embryonic serum samples were 2.34%, 5.16%, and 5.37%; and interassay variation across all three assays was determined to be 23.79%. The assay of neonate serum samples had an intrassay variation of 10.61%.
CORTICOSTERONE IN CHICK EMBRYOS n
10
II
E
13
14
15
16
17
18
19
20
21
DAYS OF INCUBATION
FIG. 1. Serum concentrations (means ± SEM) of corticosterone from developing chick embryos and neonates. Solid lines are representative of the regression relationships fitted to the data.
since the intent of staging the embryos was to select them effectively on the basis of similarity of development. The levels of corticosterone were examined within each half of the incubation period covered in this study since previous work (Woods et al, 1971; Wise and Frye, 1973; Kalliecharan and Hall, 1974, 1976) indicates that the adrenal glands are autonomous in their secretion of corticosterone prior to about 15 to 16 days of incubation and under the control of the adenohypophysis thereafter. Serum corticosterone levels for days 10 through 16 of incubation, constituting the first half of the period studied, were different (P< .01), and a trend analysis revealed that a quadratic relationship existed for the levels of corticosterone. From the data, a quadratic function was calculated for the mean corticosterone of days 10 through 16 with the following equation:
discussed by Woods et al. (1971), Wise and Frye (1973), and Kalliecharan and Hall (1974, 1976). The pattern of corticosterone secretion reported in this study for the period of time from 10 through 16 days of incubation is similar to those reported by Wise and Frye (1973), Kalliecharan and Hall (1974), and Siegel and Gould (1976), who all measured plasma corticosterone with a competitive protein binding assay. During the time from 10 to 16 days of incubation, Wise and Frye (1973) reported plasma corticosterone values that were approximately one-fourth of those reported here. Kalliecharan and Hall (1974) reported levels approximately tenfold higher, and Siegel and Gould (1976) also graphically showed plasma corticosterone concentrations to be higher than the values of this study. Girouard and Hall (1973) reported that the mitotic activity of the adrenal gland is greatest on day 13 of incubation, and Hall (1970) stated that mitotic activity in the embryonic adrenal gland peaked on day 14. In light of these observations, the increasing presence of corticosterone in the serum of the embryos sampled from 14 to 16 days may be reflective of the increased number of secretory cells in the adrenal cortex at 13 or 14 days of incubation. The increasing secretory activity of the embryonic adrenal gland has been documented by Kalliecharan and Hall (1976), and the peak concentration of total corticosteroids in adrenal homogenates at 15 days of incubation was noted to correspond to the rapid synthesis and secretion of steroids through possible stimulation of the adrenals by the pituitary gland. Statistical analysis revealed no differences among the corticosterone values from 15 through 20 days of incubation (Table 1), and a linear relationship was fitted to the data for graphic presentation (Fig. 1). This linear relationship was calculated from the following equation:
q = 29.486 - 4.55D ; + .196D? q = - 2 . 5 7 2 + .549D; where C; = corticosterone level for the ith day, Dj = ith day, i = 10 to 16. This is graphically depicted in Figure 1, which shows that the levels fluctuate slightly from 10 to 14 days and then increase until day 16. In and around the 15th or 16th day of incubation, the secretion of corticosterone is possibly brought under control by the establishment of the adenohypophyseal-adrenal axis, which has been
where C; and Dj correspond to the designations given them for the previous equation and i = 15 to 20. Values reported here for the period of time from 15 through 20 days of incubation averaged 7.04 ng/ml. Wise and Frye (1973) found corticosterone levels one-third lower, but Kalliecharan and Hall (1974) reported values about threefold higher in embryonic
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Wise and Frye (1973) believed that increasing plasma corticosterone levels prior to hatching were the result of a hyperactive adrenal or lowered turnover rate. These researchers and Siegel and Gould (1976) indicated that it is necessary for plasma corticosterone levels to increase prior to the onset of hatching for duodenal epithelium differentiation, and for increases in alkaline phosphatase levels which were studied by Moog and Richardson (1955). Kalliecharan and Hall (1974) believed that a decrease in corticosteroid concentrations in the embryonic plasma prior to hatching is representative of increased hormone metabolism, decreased synthesis, and decreased secretion at that time. Corticosterone binding capacity (Siegel and Gould, 1976; Martin et al., 1977; Gasc and Martin, 1978) has been shown to decrease after 15 or 16 days of incubation allowing for more circulating free or active corticosterone perhaps necessary for the onset of hatching. However, if the amount of corticosterone (or glucocorticoids) present is too great, delayed hatching time, decreased hatchability,
and improper yolk sac retraction will occur (Brooks and Ungar, 1967; Wishart et al, 1977). When the chick neonate hatches, the presence of large concentrations of circulating corticosterone and other glucocorticoids are suspected since this initial emergence is believed to be quite eventful for the chick. A graphic representation of the mean levels of corticosterone determined from a group of chicks ranging up to 12 hr posthatch in age is shown in Figure 2. In order to show the degree of variation (possibly due to episodic release) associated with sampling chicks within an hourly interval, the four 15-min sampling periods were included. Analysis of these data indicated no differences existed among any of the hatching groups or the sampling periods. This indicates that within a 12 hr posthatch time range, blood sampling for corticosterone determination can be done on a group of neonate chicks without detection of differences in corticosterone levels if hatching time has been disregarded. Gildersleeve et al. (1978) and Satterlee et al. (1980) used the radioimmunoassay procedure employed in the present study to measure the first 24 hr fluctuations of plasma corticosterone in commercially obtained broiler chicks which had been debeaked and vaccinated at the
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P2 P3 P4 PI P2 P3 P4 PI P2 P3 P4 PI P2 P3 P4 HI H2 H3 H4
HATCHING TIMES (H) AND SAMPLING PERIODS (P)
FIG. 2. Serum concentrations (mean ± SEM) of corticosterone from neonate chicks. Hatching times are as follows: HI = 10 to 12 hr posthatch, H2 = 7 to 9 hr posthatch, H3 = 4 to 6 hr posthatch, H4 = 1 to 3 hr posthatch. Sampling periods are as follows: PI = first 15 min of hour, P2 = second IS min of hour, P3 = third 15 min of hour, P4 = fourth 15 min of hour.
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plasma. Siegel and Gould (1976) also found higher levels than those presented here, and the only other measure of corticosterone with a radioimmunoassay (Nakamura et al., 1978) found 17-day-old embryos to have levels of 7.20 ng/ml. No differences among serum corticosterone concentrations from 15 through 20 days of incubation may be indicative of the increasing divergence among embryos in preparation for hatching, or perhaps there is no reason for serum corticosterone levels to increase significantly prior to hatching. Although the day 15 through 20 corticosterone values did not vary statistically, an upwards trend to day 20 was apparent. Indeed, the serum corticosterone value at day 20 was elevated 39% over the day 15 value. A comparison of the standard errors associated with the corticosterone means in both periods of the incubation time studied here reveals that more variation is found during that time when the adenohypophysis is believed to be influencing corticosterone secretion. The assumption that the adrenal gland is autonomous in its secretion of corticosterone prior to day 15 or 16 has already been made; and the continuance of this autonomy with superimposed adenohypophyseal influences could perhaps explain the larger degree of variation observed during the time just before and through day 20.
CORTICOSTERONE IN CHICK EMBRYOS
ACKNOWLEDGMENTS T h e senior a u t h o r thanks A. B. Watts, J. A. Hebert, Jr., a n d E. W. Byrd, Jr., for their instruction, advice, a n d review of this m a n u script. T h e help of Stephanie G. Scott, W. T. Springer, R. J. Daigle, and Marlene Ochoa is very m u c h appreciated, and t h e a u t h o r also t h a n k s Linda J o e for t y p i n g t h e m a n u s c r i p t .
REFERENCES Betz, T. W., 1967. The effects of embryonic pars distalis grafts on the development of hypophysectomized chick embryos. Gen. Comp. Endocrinol. 9:172-186. Brooks, W. S., and F. Ungar, 1967. Effects of C „ methyl steroids on the musculus complexus and hatching of the chick. Proc. Soc. Exp. Biol. Med. 125:488-492. Case, J. F., 1952. Adrenal cortical-anterior pituitary relationships during embryonic life. Ann. NY Acad. Sci. 55:147-158. Clawson, R. C , and L. V. Domm, 1964. Growth and liver glycogen in the chick embryo under the influence of corticosteroids. Physiol. Zool. 37: 307-315. Endocrine Sciences, 1972. Plasma corticosterone radioimmunoassay procedure, antiserum B 2142. 18418 Oxnard Street, Tarzana, CA. Evans, H. J., 1953. Action of cortisone on developing chick embryo. Proc. Soc. Exp. Biol. Med. 83: 31-34. Fugo, N. W., 1940. Effects of hypophysectomy in the chick embryo. J. Exp. Zool. 85:271-297. Gasc, J. M., and B. Martin, 1978. Plasma corticosterone binding capacity in the partially decapitated chick embryo. Gen. Comp. Endocrinol. 35: 274-279. Gildersleeve, R. P., D. G. Satterlee, and W. A. Johnson, 1978. Plasma glucocorticoid levels in broiler chicks using two blood sampling techniques. Poultry Sci. 57:1138. Girouard, R. J., and B. K. Hall, 1973. Pituitary-adrenal
interactions and growth of the embryonic avian adrenal gland. J. Exp. Zool. 183:323-332. Hall, B. K., 1970. Response of host embryonic chicks to grafts of additional adrenal glands. 1. Response of host adrenal glands, gonads, and kidneys. Can. J. Zool. 48:867-872. Hall, B. K., 1971. Response of host embryonic chicks to grafts of additional adrenal glands. 3. Response of adrenal glands and gonads to the pressence of homogenates of adrenal glands. Can. J. Zool. 49:381-384. Hall, B. K., and R. Kalliecharan, 1975. The effects of exogenous cortisone acetate on development, especially skeletal development, and on circulating levels of corticosteroids in chick embryos. Teratology 12:111-119. Hamburger, V., and H. L. Hamilton, 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 8 8 : 4 9 - 9 2 . Kalliecharan, R., and B. K. Hall, 1974. A developmental study of the levels of progesterone, corticosterone, Cortisol, and cortisone circulating in plasma of chick embryos. Gen. Comp. Endocrinol. 24:364-372. Kalliecharan, R., and B. K. Hall, 1976. A developmental study of progesterone, corticosterone, Cortisol, and cortisone in the adrenal gland of the embryonic chick. Gen. Comp. Endocrinol. 30: 404-409. Karnofsky, D. A., L. P. Ridgeway, and P. A. Patterson, 1951. Growth inhibiting effect of cortisone acetate on the chick embryo. Endocrinology 48: 596-616. Martin, B., J. M. Gasc, and M. Thibier, 1977. C 21 steroid binding proteins and progesterone levels in chicken plasma during ontogenesis. J. Steroid Biochem. 8:161-166. Moog, F., and D. Richardson, 1955. The influence of adrenocortical hormones on differentiation and phosphate synthesis in the duodenum of the chick embryo. J. Exp. Zool. 130:29—56. Nakamura, T., Y. Tanabe, and H. Hirano, 1978. Evidence of the in vitro formation of Cortisol by the adrenal gland of embryonic and young chickens (Gallus domesticus). Gen. Comp. Endocrinol. 35:302-308. Pedernera, E. A., 1971. Development of the secretory capacity of the chick embryo adrenal glands. J. Embryol. Exp. Morphol. 25:213-222. Pedernera, E. A., 1972. Adrenocorticotropic activity in vitro of the chick embryo pituitary gland. Gen. Comp. Endocrinol. 19:589-590. Sames, H. L., and J. H. Leathern, 1951. Influence of desoxycorticosterone acetate and cortisone acetate on body weight of chick embryos. Proc. Soc. Exp. Biol. Med. 78:231-232. Satterlee, D. G., R. B. Abdullah, and R. P. Gildersleeve, 1980. Plasma corticosterone radioimmunoassay and levels in the neonate chick. Poultry Sci. 59:900-905. Siegel, B. V., M. J. Smith, and B. Gersd, 1957. Effects of cortisone on the developing chick embryo. Arch. Pathol. 63:562-570. Siegel, H. S., and N. R. Gould, 1976. Chick embryonic plasma proteins and binding capacity for corticosterone. Dev. Biol. 50:510-516. Wise, P. M., and B. E. Frye, 1973. Functional develop-
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h a t c h e r y . These researchers f o u n d highest levels at the h a t c h e r y (some 2 0 ng/ml) a n d 2 0 h r later (some 11 ng/ml) w i t h lowest levels (some 6 n g / m l ) at 1 0 t o 1 4 h r after receiving t h e chicks. In light of these results, t h e mean level presented for day 21 in this s t u d y (Table 1, Fig. 1) can only be assumed t o represent t h e level of corticosterone in t h e serum at t h e particular t i m e of day w h e n t h e samples were taken from t h e n e o n a t e s . If a m e a n value can be used t o represent t h e level of corticosterone of chicks hatching a t various t i m e s up t o 12 hr after first emergence, t h e n it m a y be supposed t h a t t h e activity of those chicks already h a t c h e d influences t h e levels of corticosterone of chicks j u s t h a t c h e d and hatching.
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ment of the hypothalamo-hypophyseal-adrenal cortex axis in the chick embryo, Gallus domesticus. J. Exp. Zool. 185:277-292. Wishart, G. J., J. E. A. Leakey, and G. J. Dutton, 1977. Differential effects of hormones on precocious yolk sac retraction in chick embryos fol-
lowing administration by a new technique. Gen. Comp. Endocrinol. 31:373-380. Woods, J. E., G. W. DeVries, and R. C. Thommes, 1971. Ontogenesis of the pituitary-adrenal axis in the chick embryo. Gen. Comp. Endocrinol. 17: 407-415.
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