2101

FORCED MOLTING AND SEMEN

of Electricity to Agriculture in California. The authors are indebted to Lester Fuqua and Joe Borelli for their excellent technical assistance. REFERENCES

Effects of Stress on the Corticosterone Content of the Blood Plasma and Adrenal Gland of Intact and Bursectomized Gallus Domesticus I. N I R , D . Y A M AND M. PEREK

Department of Animal Science, Faculty of Agriculture, The Hebrew University of Jerusalem, 12, Rehovot, Israel

P.O.B.

(Received for publication March 31, 1975)

ABSTRACT The response of intact and bursectomized chicks to stressful stimuli has been examined. The stressors imposed were: a. fast-acting ACTH administration; b. immersion in cold water; c. starvation. In Bursa-intact chicks the results were as follows: 1. Plasma corticosterone was increased by all stimuli. 2. Adrenal corticosterone was decreased by ACTH treatment while it was increased by immersion in cold water and by starvation. 3. Plasma glucose was increased by ACTH administration and cold water immersion and decreased by starvation of the birds. 4. Adrenal ascorbic acid concentration was not influenced by all stimuli. 5. Adrenal weights were found to be increased by ACTH and starvation treatments only. 6. Bursa weights were increased by ACTH administration. 7. A very low concentration of corticosterone was found in the Bursa of Fabricius. Bursectomized chicks differed from the intact ones in the following: 1. Plasma and adrenal corticosterone concentrations were not increased by starvation. 2. Plasma glucose increased moderately with ACTH administration. 3. Adrenal ascorbic acid was depleted by all stimuli but was not related to the corticosterone level in the adrenals and blood plasma. POULTRY SCIENCE 54: 2101-2110, 1975

INTRODUCTION

S

INCE all stressors evince responses by the adrenals, this organ has been widely surveyed. The depletion of the adrenal ascorbic acid (AAAD) or cholesterol, and the tendency of active adrenals to hypertrophy are useful tests and can be applied to the fowl. Other changes mostly resulting from the increase in adrenal activity were also used as indicators. These changes include lyin-

phoid tissue involution, particularly the Bursa of Fabricius (Frankel, 1970; Freeman, 1971a; Siegel, 1971). The rise in adrenal venous plasma corticosteroid after ACTH treatment has also been reported (Nagra et al., 1960; Frankel et al, 1967a; Taylor et al., 1970; Weis and Brand, 1974). Attempts to deplete the adrenal stores of ascorbic acid by treating intact chicks with adrenocorticotrophic hormone (ACTH) has

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Atkinson, R. L., J. W. Bradley, C. B. Schom, T. M. Terguson and W. F. Krueger, 1974. Recycling turkey hens. Poultry Sci. 53: 847-849. Carson, J. E., F. W. Lorenz and V. S. Asmundson, 1955. Semen production in the turkey male. 1.

Seasonal variation. Poultry Sci. 34: 336-343. Harper, J. A., and J. E. Parker, 1957. Changes in seasonal egg production of turkeys induced through controlled light exposure and forced moltings. Poultry Sci. 35: 967-973. Leighton, A. T., Jr., H. P. Van Krey, D. D. Moyer and L. M. Potter, 1971. Reproductive performance of force-molted turkey breeder hens. Poultry Sci. 50: 119-126. Van Krey, H. P., A. T. Leighton, Jr. and D. D. Moyer, 1967. Force molting of turkeys to obtain a second season of egg production. Poultry Sci. 46: 1331-1332.

2102

I. NIR, D. YAM AND M. PEREK

MATERIALS AND METHODS The chicks used were crossbred New Hampshire x White Leghorn males. At the age of one day, chicks were anaesthetized

with ethyl ether and then surgically bursectomized according to Chang et al. (1957). Parallel groups were sham-operated. The shamoperation consisted of an incision equal in size to the one performed in bursectomy. At autopsy the bursectomized chicks were inspected in order to ascertain that bursectomy was< complete and that no regeneration occurred. The chicks were wing-banded and kept in thermostatically controlled electrically heated batteries equipped with raised wire-mesh floors. They were fed a commercial mash containing 21% protein. All treatments: intact (I), sham-operated (S) and bursectomized (Bs) chicks were kept together. Blood was drawn by heart puncture into heparinized syringes and centrifuged. The plasma was kept at -22° C. for later determination of corticosterone and glucose. After killing by neck dislocation, the adrenals and bursa were carefully dissected, transferred to ice cold petri dishes containing saline soaked filter paper, weighed and frozen. The adrenals of each bird were homogenized in 3 ml. ice cold distilled water with a teflon pestle for the determination of ascorbic acid and corticosterone. The bursa was similarly homogenized but in 7 ml. dichloromethane for corticosterone determination. Treatments. ACTH Administration. Fastacting ACTH (Acton, Fredericksburg Chemical Lab., L.U. Copenhagen) dissolved in saline was injected in the peritoneal cavity (3 i.u./lOO g. body weight) of 21 day old chicks. Controls were injected with equal volumes of saline. The chicks were killed 1 and 3 h. following injection. 3 chicks were used per treatment and period. Their body weight was: Intact, 150 ± 10; Sham-operated, 142 ± 6and Bursectomized, 146 ± 7g. (Mean ± SEM). Immersion in Cold Water. Immersion was carried out in cold water (17° C.) on 10 day

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failed (Jailer and Boas, 1950; Elton et al., 1959; Perek and Eckstein, 1959; Freeman et al., 1966). The modification of the response of the adrenal gland to stress following bursectomy was first described by Perek and Eilat (1960) who obtained an AAAD in the bursectomized chicks. The complex relationships between the adrenals, the Bursa of Fabricius and the intensity of stress has been studied and discussed by Freeman (1967, 1969, 1970, 1971b) who advanced the hypothesis that the Bursa of Fabricius produces a ' 'factor which facilitates the maturation or perhaps the functioning of the AAAD mechanism." The stress indicators described above are observed in animals following surgery or killing; these factors by themselves are stressful stimuli which most probably affect the response to the considered stressor. This obstacle can be surmounted by assaying the peripheral blood plasma corticosterone which is the main steroid in the fowl (Phillips and Jones, 1957; Brown, 1961; Taylor et al, 1970). Franket et al. (1967b) demonstrated that the determination of blood plasma corticosterone by fluorimetric methods gave gross errors. The method used in this study utilises the steroid binding properties of corticosteroid binding globulin (CBG, transcortin). This method, developed by Murphy (1967), was shown to be rapid, specific and free from non-steroidal interference. The intent of this work was to study the influence of various stressors: ACTH administration, starving and immersion in cold water on the plasma and adrenal corticosterone, adrenal ascorbic acid and plasma glucose concentrations in intact, bursectomized and sham-operated chicks.

2103

STRESS AND CORTICOSTERONE

Starvation. Starving was carried out in 10 day old chicks during 3 days. They had free access to water. Parallel groups had free access to food and water. Eight starved and 3 fed chicks, from each treatment, were assayed 1 and 3 days following the start of starvation. Chemical Determinations. Blood plasma glucose was determined by the glucose oxydase procedure with "Biochemica Test Combination" (C.F. Boehringer und Soehne GmbH Mannheim, W. Germany). Vitamin C in adrenals was determined according to Nir et al. (1972). Corticosterone concentration in blood plasma, adrenal homogenates and bursa extracts was determined according to Murphy (1967) by the competitive protein binding radioassay. Radioactive corticosterone-Id 2 - 3 H with a specific activity of 40 Curies / mM was obtained from New England Nuclear, Mass. 02118, U.S.A. Non-radioactive corticosterone was obtained from Sigma Chemicals, U.S.A. 3 H-corticosterone-corticosteroid binding globulin solution (CBG-tracer solution) was prepared as follows: 2.5 ml. of blood plasma was drawn from a 2-4 month pregnant woman and brought to a volume of 10 ml. with distilled water and the free steroids were precipitated with 80 mg. 60-100 mesh Florisil decantated 5 times with water and activated 10 h. at 180° C. (Hopkin S. Williams Ltd., England).

The supernatant was then brought to 100 ml. with distilled water and 50-60 ng. of 3 H-corticosterone was added. The solution was kept in a refrigerator and used the next day. The determination of corticosterone in plasma and adrenals' homogenates was proceeded as follows: 0.2 ml. of plasma or of adrenal homogenates (diluted x 20-100) were transferred to 15 ml. glass-stoppered conical tubes. Five ml. of dichloromethane (Frutarom-Israel) were added, the tubes were shaken 30 seconds on an automatic shaker and then centrifuged. An aliquot of the solvent was then removed with a 5 ml. syringe fitted with a long needle. After transfer to a centrifuge tube the solvent was evaporated to dryness at 45° C. under nitrogen. Standard curves were made by adding to centrifuge tubes increasing amounts of an ethanolic solution of corticosterone and evaporating the solvent. Thereupon 1.0 ml. CBG-tracer solution was added. The tubes were shaken in a water bath (45° C.) for 5 min., then cooled in an ice cold water bath for 10 min. The absorbent was added (80 mg. florisil) with a spoon designed for this purpose and the mixture agitated 30 seconds on an automatic shaker. 0.2 ml. of the supernatant was added to 8 ml. Bray's solution: 60 g. Naphtalen (Frutarom), 200 mg. dimethyl POPOP (Packard), 4 g. PPO (Packard), 100 ml. methyl alcohol (Frutarom) and 20 ml. ethylene glycol (Riedel de Haenag-Hanover). Counting was carried out in a Packard liquid scintillation spectrometer. Corticosterone in bursa was determined on 5 ml. of the dichloromethane extract as described above. All determinations were made in triplicate.

RESULTS Effect of ACTH Administration (Fig. 1). ACTH administration caused a marked increase in the plasma corticosterone con-

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old chicks. Groups of 3 chicks from each treatment were assayed before, 5 m. and about 10 m. (at coma) after immersion. Their body weight was: Intact, 78 ± 7; Shamoperated, 79 ± 6 and Bursectomized, 77 ± 5 g. In a preliminary work it was observed that immersion of the body (head excluded) in cold water caused linear reduction in the colon temperature during 10 m., from 41° C. to about 33° C. At this temperature the chicks went into coma.

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