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Blood Cells and Chemistry of Young Chickens During Daily ACTH and Cortisol Administration
Blood Cells and Chemistry of Young Chickens During Daily A C T H and Cortisol Administration H . S. SlEGEL United States Department of Agriculture1 (Received for publication March 30, 1968)
DRENAL cortical extracts (ACE) - and glucocorticoids, as well as ACTH (adrenocorticotropin), increase acidophilic granulocytes and cause relative lymphopenia and heterophilia in chickens (Shapiro and Schechtman, 1949; Newcomer, 1957; Wolford and Ringer, 1962). However, where Shapiro and Schechtman (1949) found that ACE caused leucopenia, others have found that ACTH and cortisone acetate produced significant leucocytosis (Newcomer, 1957; Glick, 1961). Glucocorticoids and ACTH also significantly increase the levels of cholesterol and corticosterone in the plasma of fowl (Stamler, 1952; Siegel and Siegel, 1966). Adrenal steroids produce a hypercalcemia in chickens (Urist and Deutsch, 1960), and osteoperosis, as found in mammals (Storey, 1961) was demonstrated in laying hens injected with cortisone. Cortisone acetate also inhibits chondrogenesis in the long bones of cockerels (Huble, 1957). Although ACTH has been shown to lower serum calcium in rabbits (Natelson et al., 1965), its effect on the blood calcium of avian species is not clear. Previous work with chickens has shown that maximum depletion of adrenal cholesterol occurs 12 hours after a single I. M. injection of ACTH, but that the maximum plasma cholesterol and glucose responses may occur from 12 to 24 hours after administration (Siegel and Beane, 1961). It has also been shown that significant plasma
A
1
Southeast Poultry Research Laboratory, Animal Husbandry Research Division, ARS, Athens, Ga. 30601.
cholesterol responses to ACTH do not appear before three weeks of age in chickens, but that responses to Cortisol may be observed during the first week (Siegel, 1962; Siegel and Siegel, 1966). This paper describes the daily changes in packed cell volume (PCV), white blood cells (WBC), cholesterol, corticosterone and calcium in the blood of young Athens Randombred chickens over a four-day period of continuous Cortisol and ACTH administration. EXPERIMENTAL METHODS
A total of 96 four-week-old male Athens Randombred chickens was divided equally into six treatment groups as follows: 1. ACTH2—1 U.S.P. unit per 100 g. body weight per day 2. ACTH2—2 U.S.P. units per 100 g. body weight per day 3. Cortisol3—0.25 mg per 100 g. body weight per day 4. Cortisol3—0.50 mg per 100 g. body weight per day 5. Bled control—gelatin-injected 6. Non-bled control—gelatin-injected All birds were given daily intramuscular injections into the pectoral area. The ACTH was in a 15% (W/V) gelatin vehicle, the Cortisol in 0.85% NaCl, and the two control groups received only 15% gelatin. 2
ACTH—Adrenocorticotropin—Depo - ACTH, the Upjohn Company. 3 Cortisol (hydrocortisone)—A4 = pregene—11|3, 17a, 21—triol 3, 20— dione— Cortef, The Upjohn Company.
1811
1812
H . S. SlEGEL TABLE 1.-
-White blood cells, packed cell volumes and plasma chemistry of held and non-bled1 A Ihens randombred chickens after four days of gelatin injections
Variable White Blood Cells Total WBC/mm. 3 (X103) Basophils, /mm. 3 (X103) Eosinophils, /mm. 3 (X103) Heterophils, /mm. 3 (X103) Lymphocytes, /mm. 3 (X103) Monocytes, /mm. 3 (X103) P.C.V. % Plas. Cholesterol, mg.% Plas. Corticosterone, pg.% Plas. Calcium, mg.%
Bled
Non-bled
32.16 + 2.89 2 1.72 + 0.05 1.45 + 0.34 7.03 + 2.45 25.41 + 2.27 0.98±0.29 24.1 + 1.4 239+19 10.6+1.0 8.8 + 0.9
32.92 + 3.48 2 1.64 + 0.17 1.75 + 0.66 3.83 + 1.40 28.31 + 3.16 1.46 + 0.31 27.7 + 0.8** 232 + 12 12.8+1.2 9.2±0.9
1
Bled—3 ml. blood sample taken daily. Non-bled- -3 ml. blood sample taken only on final day. Means of 16 birds+ SE. **p<0.01. 2
Each day, for five days, a three ml. sample of blood was taken from the brachial vein with heparinized syringes (alternately from the left and right wings) from each bird of each treatment group, except the non-bled controls. Samples were taken from the latter only on the last day. Immediately after each day's sampling, the daily treatments were injected intramuscularly. The first day's sample acted as a baseline sample for each bird and was designated as 0 day, with succeeding samples designated as days 1, 2, 3 and 4. The non-bled controls were compared with the bled controls on day 4 to determine the effect of daily bleeding, per se, on the measured variables. Blood cell and chemical determinations were made as follows: Duplicate PCV were determined in capillary tubes spun at approximately 12,000 X g. for five minutes (Cohen, 1967). Total WBC were counted in duplicate in a hemocytometer, using a Rees and Ecker (1923) stain. Differential white blood cell counts, in which 300 cells per slide were counted, were made from Wright's stained smears (Lucas and Jamroz, 1961). The remainder of the sample was centrifuged and the plasma stored at — 20°C. until analyzed. Plasma cholesterol was de-
termined by the method of Zlatkis et al. (1953), corticosterone, or more properly sulfuric acid fluorogens (Frankel et al., 1967), were determined by the method described by Guillemin et al. (1959); and calcium was estimated fluorometrically by the Wallach and Steck procedure described in the Manual of Fluorometric Clinical Procedures (G. K. Turner, Assoc, 1964). All determinations were done in duplicate, and where more than 5% separated duplicates, the determinations were repeated. Preliminary analyses of variance were computed to determine the significance of the differences between bled and non-bled controls after four days of gel injection. Following this, analyses of variance using a split-plot design were calculated with the non-bled control group deleted. Where treatments within a day differed significantly, Kramer's (1956) modified multiple range test was applied to separate the means. RESULTS AND DISCUSSION
The mean values for bled and non-bled controls, which are shown in Table 1, indicated that daily removal of three ml. of blood for four days resulted in significantly lower PCV. The effect of blood sampling on PCV has been discussed (Sturkie and
EFFECTS OF
ACTH
1813
AND CORTISOL
TABLE 2.—Analyses of variance of daily changes in total white cells and absolute white blood cell counts during four days of ACTH and Cortisol injections to Athens randombred males M e a n Squares
Total Days of Treatment
%
Counts/mm. 3
Soura ; of Variation
Eosinophil Heterophil Lyr mphocytei Monocyte
PCV
DF
Total W B C
Basophil
(X10»
(X10*)
(X10>)
(X10»)
(X10=)
(X10<)
394 4
3,943**
299**
363**
419**
166**
1601**
416**
0
Treatment birds w / n day
4 74
433 217
77 101
5 19
3 191
89 40
146 179
2 38
1
Treatment birds w / n d a y
4 74
150 203
IS 12
38 32
1 15
43 39
17 19
11 8
2
Treatment birds w / n day
4 73
2,196** 133
62* 24
20 18
16 13
64** 25
19 51
12 7
3
Treatment birds w / n d a y
4 75
553* 185
23 37
12 35
56* 22
166** 42
71 38
17 12
4
Treatment birds w / n day
4 74
681* 246
43 36
11 23
135* 53
190** 53
33 104
4 9
197
42
25
59
78
15
Pooled Error
370
40
*P<0.05. **P<0.01.
Newman, 1951). Differential counts, total WBC and the three plasma compounds measured were not significantly affected by the bleeding procedures per se. Blood cells. The split-plot analyses of daily changes in PCV, WBC and differential white cell counts are shown in Table 2 and the mean values are shown in Figures 1, 2, and 3. The pooled errors were used to test differences among days, while birds within days were used to test differences among treatments within days. PCV's of all treatment groups declined in essentially a linear fashion with days of treatment (Figure 1). This was related to the sampling procedure since non-bled control values were significantly higher than bled controls at the end of the experiment. Neither Cortisol nor ACTH, at the levels used, significantly influenced the PCV. The total WBC counts of all treatment groups declined slightly on the second day of treatment but were above initial values by the fourth day (Figure 1). Differences between treatments were not observed until the second day. At this time, a significant leucopenia was observed with 2 units of ACTH and 0.50 mg. of Cortisol/lOOg./day
which continued until termination of the experiment. These results agree with those of Shapiro and Schechtman (1949) and suggest that the leucocytosis described by Newcomer (1957) and Glick (1961) with single dosages may have been transient. The responses of the various polymorphonuclear cells are shown in Figure 2. Basophil counts of birds receiving 0.50 mg. of Cortisol per day were significantly lower only on the second day of treatment, while eosinophils were not significantly changed throughout the four-day period. Heterophil counts increased during the experiment and by the third day counts were significantly higher than controls in birds given 0.50 mg. cortisol/lOOg./day. Changes in the numbers of mononuclear cells over the experimental period are shown in Figure 3. Monocyte counts were generally lower during the first three days, but returned to or above initial values by the fourth day. No significant treatment differences were observed. The numbers of lymphocytes in the bled controls declined on day 2 and then increased on the third and fourth days. Significant lymphopenia, which occurred in the 0.50 mg Cortisol-
1814
H . S. SlEGEL
o non-Wed
ftACTH
1U
\ ACTH 2U
o non-bled pACTH 1U acort .25 / .' 4cort .50 /.* *ACTH2U
were significant day effects which were primarily due to the treatment effect on succeeding days of treatment (i.e., a day X treatment interaction). Plasma corticosterone, measured as sulfuric acid fluorogens, was significantly higher than controls in both cortisol-treated groups after three days of treatment, and in the 2 unit ACTH group by the fourth day (Figure 4 ) . In a paper published after our determinations were made, Frankel et al. (1967) questioned the validity of rapid fluorometric methods for the estimation of corticosterone due to the possible interference of other fluorogens, particularly in highly lipemic avian blood. Because Cortisol and ACTH induce hyperlipemia, especially cholesteremia (Drury, 1959; Siegel, 1962; see also below), it is possible that this may have caused the observed "corticosterone" effect. Cholesterol levels in the blood were sig-
DAY FIG. 1. Effect of daily administrations of ACTH and Cortisol on packed cell volume (PCV) and total white blood cells (WBC). Means within days covered by the same vertical line do not differ significantly. ® Non-bled control, • Bled control, • ACTH-1 units, • ACTH-2 units, A Cortisol-0.25 mg., • Cortisol-0.50 mg.
treated group by the second day, continued through the fourth day. On the third and fourth days, the values for all treated groups were significantly below the control value. These results agree with those of others (Sturkie, 1965), and since lymphocytes make up approximately 8 0 % of the white blood cells (Lucas and Jamroz, 1961), this would account for the observed leucopenia. Blood Chemistry. In Table 3 are shown the split-plot analyses and in Figure 4 the graphic representation of the effects of ACTH and Cortisol on the three plasma compounds measured. In all three, there
ort.25
FIG. 2. Effect of daily administrations of ACTH and Cortisol on polymorphonuclear white blood cells. Symbols the same as Fig. 1.
EFFECTS OF
ACTH
nificantly higher in the two cortisol-injected groups after one day of treatment, and by the second day, those receiving 2 units of ACTH also had significantly higher levels than controls. Values for the treated birds plateaued at higher levels by about the second day of treatment for the cortisoltreated birds. These results are in agreement with previous work (Drury, 19S9; Siegel, 1962). Plasma calcium concentrations of groups receiving 0.50 mg. of Cortisol were significantly higher than control values on the third and fourth day of treatment. ACTH at the levels used did not have a significant influence on plasma calcium. The hypercalcemic response which is apparently the result of increased bone resorption by osteo-
E
*
2 cort.5CT
5 1
-I
^^**$0£>
-cort.25 ITACTH 1U •V,vACTH.2U non-ble led -non-bed
26r
1815
AND CORTISOL
clasts (Storey, 1961) required a relatively high dose. In previous work cortisone acetate at 1.4 mg./lOOg. body/day was shown to inhibit chondrogenesis slightly in chickens (Huble, 1957). Unlike the response in rabbits, where ACTH in small quantities depressed serum calcium (Natelson et al., 1965), little effect of ACTH was observed in these birds. Of importance in this experiment was the difference in the timing of response by the measured variables. The most prominent and permanent white cell response was by the lymphocytes. Basophilic changes were transient and a significant increase in heterophils was only observed with the higher level of Cortisol. Hypercholesteremia, the most rapid of the responses measured, is part of the general increase in lipid mobilization which is due to an alteration in the lipid transport mechanism in glucocorticoid or ACTHtreater animals (Drury, 1959; Jaussi et al., 1962). The plateauing of cholesterol and sulfuric acid fluorogens after three days of treatment suggests a metabolic adjustment to the hormonal stimulation. SUMMARY
ACTH 2U
DAY Fie. 3. Effect of daily administrations of ACTH and Cortisol on mononuclear white blood cells. Symbols the same as Fig. 1.
Daily blood samples were taken from four-week-old Athens randombred chicks which were receiving slow-release forms of Cortisol and ACTH over a five-day period. Significantly lower total white blood cell counts were observed after two days of injection with either 2 U.S.P. units of ACTH or 0.50 mg. of cortisol/lOOg. body wt./day. This leucopenia was related to the lymphopenia observed at the same time. A reduction in basophils on the second day of treatment with 0.5 mg. of Cortisol was transient and differences from controls were not significant on the third or fourth day. Heterophils were significantly increased by 0.5 mg. Cortisol on the third and fourth day of treatment. Plasma cholesterol of groups re-
1816
H . S. SlEGEL TABLE 3.—Analyses of variance of daily changes in plasma total cholesterol and total calcium during 4-day administration of ACTH and Cortisol Corticosterone
Source of Variation Total Days of Treatment 0 Treatment birds w/n dav 1 Treatment birds w/n day 2 Treatment birds w/n day 3 Treatment birds w/n day 4 Treatment birds w/n day Pooled Error
Cholesterol
DF
MS
DF
368 4 4 62 4 71 4 70 4 69 4 72 344
— 313.7** 22.5 33.1 14.9 13.4 18.0 22.5 31.1* 8.6 64.8** 16.9 18.5
385 4 4 74 4 73 4 71 4 69 4 74 361
MS (X10 2 ) 1,486** 26 15 224** 17 797** 28 662** 49 950** 64 35
Calcium DF
MS
392 4 4 72 4 74 4 74 4 74 4 74 368
37.07* 4.29 4.02 13.96* 5.19 6.87 8.04 13.17* 5.25 6.75* 2.70 5.04
..
*p<0.05. **p<0.01.
ceiving 0.25 and 0.50 mg. of Cortisol rose significantly on the first day of treatment, continued to rise until the third day and then plateaued at a new higher level. In birds receiving 2 U.S.P. units of ACTH,
significant cholesteremia was not found until the second day of treatment. Corticosterone determined as the sulfuric acid fluorogens, became significantly higher than controls by the third day of treatment in groups receiving 0.S0 mg. and 0.2S mg. of Cortisol and by the fourth day in groups receiving 2 units of ACTH. Plasma calcium values were significantly elevated on the third and fourth days of treatment only in groups receiving 0.50 mg. cortisol/100g./ day. ACKNOWLEDGMENTS The author wishes to thank Mrs. Dianne Bumpus, Mr. Sam Henderson and Miss Sara McCurley for their aid during various portions of this study.
CThC-ACTh
,u
REFERENCES Cohen, R. R., 1967. Anticoagulation, centrifugation time, and sample replicate number in the microhematocrit method for avian blood. Poul-
non bled :gel
-\ row Fie. 4. Effect of daily administrations of ACTH and Cortisol on plasma calcium, cholesterol and sulfuric acid fluorogens (corticosterone). Symbols the same as Fig. 1.
EFFECTS OF
ACTH
try Sci. 46: 214-218. Drury, A., 1959. Influence of cortisone in lipid distribution and atherogenesis. Ann. New York Acad. Sci. 72 : 870-884. Frankel, A. I , B. Cook, J. W. Graber and A. V. Xalbandov, 1967. Determination of corticosterone in plasma by fluorometric techniques. Endocrinology, 80: 181-194. Glick, B., 1961. The effect of bovine growth hormone, desoxycorticosterone acetate and cortisone acetate on the white blood cell counts of 2-week-old chickens. Poultry Sci. 40: 1537-1539. Guillemin, R., G. W. Clayton, H. S. Lipscomb and S. D. Smith, 1959. Fluorometric measurement of rat plasma and adrenal corticosterone concentration. J. Lab. Clin. Med. 53 : 830-832. Huble, J., 1957. Effects of cortisone acetate on chondrogenesis and ossification on cockerels. Acta Endocrinol. 25 : 59-63. Jaussi, A. W., W. S. Newcomer and R. H. Thayer, 1962. Hyperlipemic effect of ACTH injections in the chicken. Poultry Sci. 4 1 : 528-532. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics, 12: 307-310. Lucas, A. M., and C. Jamroz, 1961. Atlas of Avian Hematology. Agriculture Monograph 25, United States Department of Agriculture. Natelson, S., G. Rannazzisi and J. B. Pincus, 1965. Effect of ACTH and vasopressin on serum calcium and citrate levels in the rabbit. Endocrinology, 77: 108-113. Newcomer, W. S., 1957. Blood cell changes following ACTH injection in the chick. Proc. Soc. Exp. Biol. Med. 96: 613-616. Rees, M., and E. E. Ecker, 1923. An improved method for counting blood platelets. J. Am. Med. Assoc. 80: 621-622. Shapiro, A. B., and A. M. Schechtman, 1949. Ef-
AND CORTISOL
1817
fect of adrenal cortical extract on the blood picture and serum proteins of fowl. Proc. Soc. Exp. Biol. Med. 70: 440-445. Siegel, H. S., and W. L. Beane, 1961. Time responses to single intramuscular doses of ACTH in chickens. Poultry Sci. 40: 216-219. Siegel, H. S., 1962. Age and sex modification of responses to adrenocorticotropin in young chickens. 2. Changes in adrenal cholesterol and blood constituent levels. Poultry Sci. 4 1 : 321-334. Siegel, H. S., and P. B. Siegel, 1966. Genetic variation in responses to repeated administrations of ACTH and hydrocortisone in immature chickens. Poultry Sci. 45: 901-912. Stamler, J., 1952. Effects of adrenal steroid compound F in depancreatized, cholesterol-fed cockerels. Fed. Proc. 11: 153. Storey, E., 1961. Cortisone induced bone resorption in the rabbit. Endocrinology, 68: 533-542. Sturkie, P. D., and H. J. Newman, 1951. Plasma proteins of chickens as influenced by time of laying, ovulation, number of blood samples taken and plasma volume. Poultry Sci. 30: 240-248. Sturkie, P. D., 1965. Avian Physiology. Cornell University Press, Ithaca, N.Y. Turner, G. K. Associates, 1964. Manual of Fluorometric Clinical Procedures. Palo Alto, Calif. Urist, M. R., and N. M. Deutsch, 1960. Effects of cortisone upon blood, adrenal cortex, gonads, and the development of osteoporosis in birds. Endocrinology, 66: 805-818. Wolford, J. H., and R. K. Ringer, 1962. Adrenal weight, adrenal ascorbic acid, adrenal cholesterol and differential leucocyte counts as physiological indicators of "stressor" agents in laying hens. Poultry Sci. 4 1 : 1521-1529. Zlatkis, A., B. Zak and A. J. Boyle, 1953. A new method for direct determination of serum cholesterol. J. Lab. Clin. Med. 4 1 : 486-492.
NEWS AND NOTES (continued from page 1810) will be accepted. If limitation should become words. It is expected that these limitations will necessary, preference will be given to those aucontinue to prevail. thors who will attend the Congress. The following references are suggested as guides in presenting the papers: If a paper is accepted but the author cannot 1. Rules for acceptance of papers presented by present the paper, it is the author's responsibility Australian Scientific Program Committee, Poultry to designate a person qualified to present the Sci. 40:553, 1961. paper. 2. Rules for competition of Poultry Science For past Congresses, papers have been limited to 2500 words with abstracts limited to 300 travel grant, Poultry Sci. 40:554, 1961. (continued on page 1921)
DRENAL cortical extracts (ACE) - and glucocorticoids, as well as ACTH (adrenocorticotropin), increase acidophilic granulocytes and cause relative lymphopenia and heterophilia in chickens (Shapiro and Schechtman, 1949; Newcomer, 1957; Wolford and Ringer, 1962). However, where Shapiro and Schechtman (1949) found that ACE caused leucopenia, others have found that ACTH and cortisone acetate produced significant leucocytosis (Newcomer, 1957; Glick, 1961). Glucocorticoids and ACTH also significantly increase the levels of cholesterol and corticosterone in the plasma of fowl (Stamler, 1952; Siegel and Siegel, 1966). Adrenal steroids produce a hypercalcemia in chickens (Urist and Deutsch, 1960), and osteoperosis, as found in mammals (Storey, 1961) was demonstrated in laying hens injected with cortisone. Cortisone acetate also inhibits chondrogenesis in the long bones of cockerels (Huble, 1957). Although ACTH has been shown to lower serum calcium in rabbits (Natelson et al., 1965), its effect on the blood calcium of avian species is not clear. Previous work with chickens has shown that maximum depletion of adrenal cholesterol occurs 12 hours after a single I. M. injection of ACTH, but that the maximum plasma cholesterol and glucose responses may occur from 12 to 24 hours after administration (Siegel and Beane, 1961). It has also been shown that significant plasma
A
1
Southeast Poultry Research Laboratory, Animal Husbandry Research Division, ARS, Athens, Ga. 30601.
cholesterol responses to ACTH do not appear before three weeks of age in chickens, but that responses to Cortisol may be observed during the first week (Siegel, 1962; Siegel and Siegel, 1966). This paper describes the daily changes in packed cell volume (PCV), white blood cells (WBC), cholesterol, corticosterone and calcium in the blood of young Athens Randombred chickens over a four-day period of continuous Cortisol and ACTH administration. EXPERIMENTAL METHODS
A total of 96 four-week-old male Athens Randombred chickens was divided equally into six treatment groups as follows: 1. ACTH2—1 U.S.P. unit per 100 g. body weight per day 2. ACTH2—2 U.S.P. units per 100 g. body weight per day 3. Cortisol3—0.25 mg per 100 g. body weight per day 4. Cortisol3—0.50 mg per 100 g. body weight per day 5. Bled control—gelatin-injected 6. Non-bled control—gelatin-injected All birds were given daily intramuscular injections into the pectoral area. The ACTH was in a 15% (W/V) gelatin vehicle, the Cortisol in 0.85% NaCl, and the two control groups received only 15% gelatin. 2
ACTH—Adrenocorticotropin—Depo - ACTH, the Upjohn Company. 3 Cortisol (hydrocortisone)—A4 = pregene—11|3, 17a, 21—triol 3, 20— dione— Cortef, The Upjohn Company.
1811
1812
H . S. SlEGEL TABLE 1.-
-White blood cells, packed cell volumes and plasma chemistry of held and non-bled1 A Ihens randombred chickens after four days of gelatin injections
Variable White Blood Cells Total WBC/mm. 3 (X103) Basophils, /mm. 3 (X103) Eosinophils, /mm. 3 (X103) Heterophils, /mm. 3 (X103) Lymphocytes, /mm. 3 (X103) Monocytes, /mm. 3 (X103) P.C.V. % Plas. Cholesterol, mg.% Plas. Corticosterone, pg.% Plas. Calcium, mg.%
Bled
Non-bled
32.16 + 2.89 2 1.72 + 0.05 1.45 + 0.34 7.03 + 2.45 25.41 + 2.27 0.98±0.29 24.1 + 1.4 239+19 10.6+1.0 8.8 + 0.9
32.92 + 3.48 2 1.64 + 0.17 1.75 + 0.66 3.83 + 1.40 28.31 + 3.16 1.46 + 0.31 27.7 + 0.8** 232 + 12 12.8+1.2 9.2±0.9
1
Bled—3 ml. blood sample taken daily. Non-bled- -3 ml. blood sample taken only on final day. Means of 16 birds+ SE. **p<0.01. 2
Each day, for five days, a three ml. sample of blood was taken from the brachial vein with heparinized syringes (alternately from the left and right wings) from each bird of each treatment group, except the non-bled controls. Samples were taken from the latter only on the last day. Immediately after each day's sampling, the daily treatments were injected intramuscularly. The first day's sample acted as a baseline sample for each bird and was designated as 0 day, with succeeding samples designated as days 1, 2, 3 and 4. The non-bled controls were compared with the bled controls on day 4 to determine the effect of daily bleeding, per se, on the measured variables. Blood cell and chemical determinations were made as follows: Duplicate PCV were determined in capillary tubes spun at approximately 12,000 X g. for five minutes (Cohen, 1967). Total WBC were counted in duplicate in a hemocytometer, using a Rees and Ecker (1923) stain. Differential white blood cell counts, in which 300 cells per slide were counted, were made from Wright's stained smears (Lucas and Jamroz, 1961). The remainder of the sample was centrifuged and the plasma stored at — 20°C. until analyzed. Plasma cholesterol was de-
termined by the method of Zlatkis et al. (1953), corticosterone, or more properly sulfuric acid fluorogens (Frankel et al., 1967), were determined by the method described by Guillemin et al. (1959); and calcium was estimated fluorometrically by the Wallach and Steck procedure described in the Manual of Fluorometric Clinical Procedures (G. K. Turner, Assoc, 1964). All determinations were done in duplicate, and where more than 5% separated duplicates, the determinations were repeated. Preliminary analyses of variance were computed to determine the significance of the differences between bled and non-bled controls after four days of gel injection. Following this, analyses of variance using a split-plot design were calculated with the non-bled control group deleted. Where treatments within a day differed significantly, Kramer's (1956) modified multiple range test was applied to separate the means. RESULTS AND DISCUSSION
The mean values for bled and non-bled controls, which are shown in Table 1, indicated that daily removal of three ml. of blood for four days resulted in significantly lower PCV. The effect of blood sampling on PCV has been discussed (Sturkie and
EFFECTS OF
ACTH
1813
AND CORTISOL
TABLE 2.—Analyses of variance of daily changes in total white cells and absolute white blood cell counts during four days of ACTH and Cortisol injections to Athens randombred males M e a n Squares
Total Days of Treatment
%
Counts/mm. 3
Soura ; of Variation
Eosinophil Heterophil Lyr mphocytei Monocyte
PCV
DF
Total W B C
Basophil
(X10»
(X10*)
(X10>)
(X10»)
(X10=)
(X10<)
394 4
3,943**
299**
363**
419**
166**
1601**
416**
0
Treatment birds w / n day
4 74
433 217
77 101
5 19
3 191
89 40
146 179
2 38
1
Treatment birds w / n d a y
4 74
150 203
IS 12
38 32
1 15
43 39
17 19
11 8
2
Treatment birds w / n day
4 73
2,196** 133
62* 24
20 18
16 13
64** 25
19 51
12 7
3
Treatment birds w / n d a y
4 75
553* 185
23 37
12 35
56* 22
166** 42
71 38
17 12
4
Treatment birds w / n day
4 74
681* 246
43 36
11 23
135* 53
190** 53
33 104
4 9
197
42
25
59
78
15
Pooled Error
370
40
*P<0.05. **P<0.01.
Newman, 1951). Differential counts, total WBC and the three plasma compounds measured were not significantly affected by the bleeding procedures per se. Blood cells. The split-plot analyses of daily changes in PCV, WBC and differential white cell counts are shown in Table 2 and the mean values are shown in Figures 1, 2, and 3. The pooled errors were used to test differences among days, while birds within days were used to test differences among treatments within days. PCV's of all treatment groups declined in essentially a linear fashion with days of treatment (Figure 1). This was related to the sampling procedure since non-bled control values were significantly higher than bled controls at the end of the experiment. Neither Cortisol nor ACTH, at the levels used, significantly influenced the PCV. The total WBC counts of all treatment groups declined slightly on the second day of treatment but were above initial values by the fourth day (Figure 1). Differences between treatments were not observed until the second day. At this time, a significant leucopenia was observed with 2 units of ACTH and 0.50 mg. of Cortisol/lOOg./day
which continued until termination of the experiment. These results agree with those of Shapiro and Schechtman (1949) and suggest that the leucocytosis described by Newcomer (1957) and Glick (1961) with single dosages may have been transient. The responses of the various polymorphonuclear cells are shown in Figure 2. Basophil counts of birds receiving 0.50 mg. of Cortisol per day were significantly lower only on the second day of treatment, while eosinophils were not significantly changed throughout the four-day period. Heterophil counts increased during the experiment and by the third day counts were significantly higher than controls in birds given 0.50 mg. cortisol/lOOg./day. Changes in the numbers of mononuclear cells over the experimental period are shown in Figure 3. Monocyte counts were generally lower during the first three days, but returned to or above initial values by the fourth day. No significant treatment differences were observed. The numbers of lymphocytes in the bled controls declined on day 2 and then increased on the third and fourth days. Significant lymphopenia, which occurred in the 0.50 mg Cortisol-
1814
H . S. SlEGEL
o non-Wed
ftACTH
1U
\ ACTH 2U
o non-bled pACTH 1U acort .25 / .' 4cort .50 /.* *ACTH2U
were significant day effects which were primarily due to the treatment effect on succeeding days of treatment (i.e., a day X treatment interaction). Plasma corticosterone, measured as sulfuric acid fluorogens, was significantly higher than controls in both cortisol-treated groups after three days of treatment, and in the 2 unit ACTH group by the fourth day (Figure 4 ) . In a paper published after our determinations were made, Frankel et al. (1967) questioned the validity of rapid fluorometric methods for the estimation of corticosterone due to the possible interference of other fluorogens, particularly in highly lipemic avian blood. Because Cortisol and ACTH induce hyperlipemia, especially cholesteremia (Drury, 1959; Siegel, 1962; see also below), it is possible that this may have caused the observed "corticosterone" effect. Cholesterol levels in the blood were sig-
DAY FIG. 1. Effect of daily administrations of ACTH and Cortisol on packed cell volume (PCV) and total white blood cells (WBC). Means within days covered by the same vertical line do not differ significantly. ® Non-bled control, • Bled control, • ACTH-1 units, • ACTH-2 units, A Cortisol-0.25 mg., • Cortisol-0.50 mg.
treated group by the second day, continued through the fourth day. On the third and fourth days, the values for all treated groups were significantly below the control value. These results agree with those of others (Sturkie, 1965), and since lymphocytes make up approximately 8 0 % of the white blood cells (Lucas and Jamroz, 1961), this would account for the observed leucopenia. Blood Chemistry. In Table 3 are shown the split-plot analyses and in Figure 4 the graphic representation of the effects of ACTH and Cortisol on the three plasma compounds measured. In all three, there
ort.25
FIG. 2. Effect of daily administrations of ACTH and Cortisol on polymorphonuclear white blood cells. Symbols the same as Fig. 1.
EFFECTS OF
ACTH
nificantly higher in the two cortisol-injected groups after one day of treatment, and by the second day, those receiving 2 units of ACTH also had significantly higher levels than controls. Values for the treated birds plateaued at higher levels by about the second day of treatment for the cortisoltreated birds. These results are in agreement with previous work (Drury, 19S9; Siegel, 1962). Plasma calcium concentrations of groups receiving 0.50 mg. of Cortisol were significantly higher than control values on the third and fourth day of treatment. ACTH at the levels used did not have a significant influence on plasma calcium. The hypercalcemic response which is apparently the result of increased bone resorption by osteo-
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-cort.25 ITACTH 1U •V,vACTH.2U non-ble led -non-bed
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AND CORTISOL
clasts (Storey, 1961) required a relatively high dose. In previous work cortisone acetate at 1.4 mg./lOOg. body/day was shown to inhibit chondrogenesis slightly in chickens (Huble, 1957). Unlike the response in rabbits, where ACTH in small quantities depressed serum calcium (Natelson et al., 1965), little effect of ACTH was observed in these birds. Of importance in this experiment was the difference in the timing of response by the measured variables. The most prominent and permanent white cell response was by the lymphocytes. Basophilic changes were transient and a significant increase in heterophils was only observed with the higher level of Cortisol. Hypercholesteremia, the most rapid of the responses measured, is part of the general increase in lipid mobilization which is due to an alteration in the lipid transport mechanism in glucocorticoid or ACTHtreater animals (Drury, 1959; Jaussi et al., 1962). The plateauing of cholesterol and sulfuric acid fluorogens after three days of treatment suggests a metabolic adjustment to the hormonal stimulation. SUMMARY
ACTH 2U
DAY Fie. 3. Effect of daily administrations of ACTH and Cortisol on mononuclear white blood cells. Symbols the same as Fig. 1.
Daily blood samples were taken from four-week-old Athens randombred chicks which were receiving slow-release forms of Cortisol and ACTH over a five-day period. Significantly lower total white blood cell counts were observed after two days of injection with either 2 U.S.P. units of ACTH or 0.50 mg. of cortisol/lOOg. body wt./day. This leucopenia was related to the lymphopenia observed at the same time. A reduction in basophils on the second day of treatment with 0.5 mg. of Cortisol was transient and differences from controls were not significant on the third or fourth day. Heterophils were significantly increased by 0.5 mg. Cortisol on the third and fourth day of treatment. Plasma cholesterol of groups re-
1816
H . S. SlEGEL TABLE 3.—Analyses of variance of daily changes in plasma total cholesterol and total calcium during 4-day administration of ACTH and Cortisol Corticosterone
Source of Variation Total Days of Treatment 0 Treatment birds w/n dav 1 Treatment birds w/n day 2 Treatment birds w/n day 3 Treatment birds w/n day 4 Treatment birds w/n day Pooled Error
Cholesterol
DF
MS
DF
368 4 4 62 4 71 4 70 4 69 4 72 344
— 313.7** 22.5 33.1 14.9 13.4 18.0 22.5 31.1* 8.6 64.8** 16.9 18.5
385 4 4 74 4 73 4 71 4 69 4 74 361
MS (X10 2 ) 1,486** 26 15 224** 17 797** 28 662** 49 950** 64 35
Calcium DF
MS
392 4 4 72 4 74 4 74 4 74 4 74 368
37.07* 4.29 4.02 13.96* 5.19 6.87 8.04 13.17* 5.25 6.75* 2.70 5.04
..
*p<0.05. **p<0.01.
ceiving 0.25 and 0.50 mg. of Cortisol rose significantly on the first day of treatment, continued to rise until the third day and then plateaued at a new higher level. In birds receiving 2 U.S.P. units of ACTH,
significant cholesteremia was not found until the second day of treatment. Corticosterone determined as the sulfuric acid fluorogens, became significantly higher than controls by the third day of treatment in groups receiving 0.S0 mg. and 0.2S mg. of Cortisol and by the fourth day in groups receiving 2 units of ACTH. Plasma calcium values were significantly elevated on the third and fourth days of treatment only in groups receiving 0.50 mg. cortisol/100g./ day. ACKNOWLEDGMENTS The author wishes to thank Mrs. Dianne Bumpus, Mr. Sam Henderson and Miss Sara McCurley for their aid during various portions of this study.
CThC-ACTh
,u
REFERENCES Cohen, R. R., 1967. Anticoagulation, centrifugation time, and sample replicate number in the microhematocrit method for avian blood. Poul-
non bled :gel
-\ row Fie. 4. Effect of daily administrations of ACTH and Cortisol on plasma calcium, cholesterol and sulfuric acid fluorogens (corticosterone). Symbols the same as Fig. 1.
EFFECTS OF
ACTH
try Sci. 46: 214-218. Drury, A., 1959. Influence of cortisone in lipid distribution and atherogenesis. Ann. New York Acad. Sci. 72 : 870-884. Frankel, A. I , B. Cook, J. W. Graber and A. V. Xalbandov, 1967. Determination of corticosterone in plasma by fluorometric techniques. Endocrinology, 80: 181-194. Glick, B., 1961. The effect of bovine growth hormone, desoxycorticosterone acetate and cortisone acetate on the white blood cell counts of 2-week-old chickens. Poultry Sci. 40: 1537-1539. Guillemin, R., G. W. Clayton, H. S. Lipscomb and S. D. Smith, 1959. Fluorometric measurement of rat plasma and adrenal corticosterone concentration. J. Lab. Clin. Med. 53 : 830-832. Huble, J., 1957. Effects of cortisone acetate on chondrogenesis and ossification on cockerels. Acta Endocrinol. 25 : 59-63. Jaussi, A. W., W. S. Newcomer and R. H. Thayer, 1962. Hyperlipemic effect of ACTH injections in the chicken. Poultry Sci. 4 1 : 528-532. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics, 12: 307-310. Lucas, A. M., and C. Jamroz, 1961. Atlas of Avian Hematology. Agriculture Monograph 25, United States Department of Agriculture. Natelson, S., G. Rannazzisi and J. B. Pincus, 1965. Effect of ACTH and vasopressin on serum calcium and citrate levels in the rabbit. Endocrinology, 77: 108-113. Newcomer, W. S., 1957. Blood cell changes following ACTH injection in the chick. Proc. Soc. Exp. Biol. Med. 96: 613-616. Rees, M., and E. E. Ecker, 1923. An improved method for counting blood platelets. J. Am. Med. Assoc. 80: 621-622. Shapiro, A. B., and A. M. Schechtman, 1949. Ef-
AND CORTISOL
1817
fect of adrenal cortical extract on the blood picture and serum proteins of fowl. Proc. Soc. Exp. Biol. Med. 70: 440-445. Siegel, H. S., and W. L. Beane, 1961. Time responses to single intramuscular doses of ACTH in chickens. Poultry Sci. 40: 216-219. Siegel, H. S., 1962. Age and sex modification of responses to adrenocorticotropin in young chickens. 2. Changes in adrenal cholesterol and blood constituent levels. Poultry Sci. 4 1 : 321-334. Siegel, H. S., and P. B. Siegel, 1966. Genetic variation in responses to repeated administrations of ACTH and hydrocortisone in immature chickens. Poultry Sci. 45: 901-912. Stamler, J., 1952. Effects of adrenal steroid compound F in depancreatized, cholesterol-fed cockerels. Fed. Proc. 11: 153. Storey, E., 1961. Cortisone induced bone resorption in the rabbit. Endocrinology, 68: 533-542. Sturkie, P. D., and H. J. Newman, 1951. Plasma proteins of chickens as influenced by time of laying, ovulation, number of blood samples taken and plasma volume. Poultry Sci. 30: 240-248. Sturkie, P. D., 1965. Avian Physiology. Cornell University Press, Ithaca, N.Y. Turner, G. K. Associates, 1964. Manual of Fluorometric Clinical Procedures. Palo Alto, Calif. Urist, M. R., and N. M. Deutsch, 1960. Effects of cortisone upon blood, adrenal cortex, gonads, and the development of osteoporosis in birds. Endocrinology, 66: 805-818. Wolford, J. H., and R. K. Ringer, 1962. Adrenal weight, adrenal ascorbic acid, adrenal cholesterol and differential leucocyte counts as physiological indicators of "stressor" agents in laying hens. Poultry Sci. 4 1 : 1521-1529. Zlatkis, A., B. Zak and A. J. Boyle, 1953. A new method for direct determination of serum cholesterol. J. Lab. Clin. Med. 4 1 : 486-492.
NEWS AND NOTES (continued from page 1810) will be accepted. If limitation should become words. It is expected that these limitations will necessary, preference will be given to those aucontinue to prevail. thors who will attend the Congress. The following references are suggested as guides in presenting the papers: If a paper is accepted but the author cannot 1. Rules for acceptance of papers presented by present the paper, it is the author's responsibility Australian Scientific Program Committee, Poultry to designate a person qualified to present the Sci. 40:553, 1961. paper. 2. Rules for competition of Poultry Science For past Congresses, papers have been limited to 2500 words with abstracts limited to 300 travel grant, Poultry Sci. 40:554, 1961. (continued on page 1921)