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Adrenal Weight, Adrenal Ascorbic Acid, Adrenal Cholesterol and Differential Leucocyte Counts as Physiological Indicators of “Stressor” Agents in Laying Hens12
STRENGTH OF VITELLINE MEMBRANE
SUMMARY
A rapid method for the evaluation of the vitelline membrane strength of the intact egg yolk was reported and evaluated. This technique involves the placement of a 2 mm. capillary tube against the vitelline membrane, the creation of a given vacuum
in the capillary tube and measurement of the time required to rupture the membrane. The time required for rupture is plotted relative to a standard curve in order to equate it with time required at other vacuums or the vacuum required to rupture the vitelline membrane in one second. It was found that temperature of the egg yolk is a factor which affects the relative strength of the vitelline membrane as measured by the capillary-vacuum method. REFERENCES Haugh, R. R., 1933. A new method for determining the quality of an egg. U.S. Egg Poultry Mag. 39 : 27, 49. Moran, T., and H. P. Hale, 1936. Physics of the hen's egg. I. Membranes in the egg. J. Exp. Biol. 13: 3S-40. Moran, T., 1936. Physics of the hen's egg. II. The bursting strength of the vitelline membrane. J. Exp. Biol. 13 : 41^17. Munro, S. S., and G. Robertson, 1935. A method of measuring the strength of the yolk membrane. U.S. Egg Poultry Mag. 4 1 : 48, 50. Sharp, P. F., 1929. What one week may do to an egg. U.S. Egg Poultry Mag. 35: 14-17.
Adrenal Weight, Adrenal Ascorbic Acid, Adrenal Cholesterol and Differential Leucocyte Counts as Physiological Indicators of "Stressor" Agents in Laying Hens1,2 Department
JOHN H. WOLFORD AND ROBERT K. RINGER of Poultry Science, Michigan State University, East Lansing, Michigan (Received for publication February 19, 1962)
T is thought that the adrenal cortex assumes an important role in an animal's ability to overcome environmental influences. Therefore, assays capable of in-
I
1 Journal Article No. 2942, Michigan Agricultural Experiment Station. 2 Part of a doctoral dissertation submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree.
dicating adrenal cortical function following changes in management, feed, lighting schedules and weather, among other factors, would possibly be a step toward the elimination of production decreases. Research with rats and guinea pigs (reviewed by Sayers, 1950) has shown that depletion of adrenal ascorbic acid, cholesterol and lipid contents occurs following an
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degree of relative elasticity nor grams pressure required to rupture the vitelline membrane could be secured. The use of a 2 mm. capillary tube was found to be preferable since a large number of measurements could be made on each intact vitelline membrane before the yolk lost its physical identity. In addition to the comparable results acquired relative to the other physical methods described in this report, the general principle of the capillary-vacuum method for evaluating the strength of the vitelline membrane may also be used in securing small samples of the egg contents from a specific yolk area for microanalyses without disrupting the general physical nature of the egg yolk.
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J . H . WOLFORD AND R . K . RlNGEE EXPERIMENTAL PROCEDURE
S. C. \Vhite Leghorn females in production for more than 40 weeks and housed either on a conventional floor or in cages were used in this study. All birds were given feed and water ad libitum, except where noted in a particular experiment. All hens designated as layers had partially formed eggs in their oviducts or had several large follicles in their ovaries at the time of sacrifice for adrenal assay. The ovaries of those birds designated as non-layers were atresic and contained only undeveloped follicles. Adrenal ascorbic acid was determined by the method of Roe and Kuether (1943), and adrenal cholesterol was determined by the method of Knobil et al. (1954). In all cases only the right adrenal gland was utilized for either the ascorbic acid or cholesterol determination. The left adrenal gland was fixed in formalin and strained with Oil Red 0 to determine the amount of neutral fat present Lillie (1954).3 These stained sections were then scored on the basis of 1 and 2 = low; 3 and 4 = medium; and 5 and 6 = high neutral fat concentration. Colorimetric readings for cholesterol and ascorbic acid were taken on a "Universal Model 14" spectrometer. Prior to sacrificing by cervical dislocation, blood samples and smears4 were taken. The blood smears were stained with Wright's stain, and a differential leucocyte count was made. Following cervical dislocation, the birds were weighed and each adrenal gland, right and left,5 was immediately removed and weighed. The data obtained were statistically ana3
The left adrenal gland was not removed in the ascorbic acid experiment. 4 Blood samples and smears were not taken in the ascorbic acid experiment. 5 The left adrenal gland was not removed in the ascorbic acid experiment.
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injection of pituitary adrenotropic hormone and following stressful conditions such as excessive muscular work or a non-fatal hemorrhage. Reports involving "stressor" agents and their influence on the avian adrenal have been more inconsistent than the reported influence on the mammalian adrenal. Howard and Constable (1958) and Siegel and Beane (1961) produced adrenal cholesterol depletion in chickens following ACTH injection; however, Elton et al. (1959) reported that adrenal cholesterol remained unchanged following ACTH and cold (0-5°C.) treatment. Perek and Eckstein (1959) were able to elicit adrenal ascorbic acid depletion in laying hens following ACTH injection. Adrenal hypertrophy, another measure of interrenal activity has been shown in male chickens by Brown et al. (1958), in quail by Zarrow and Baldini (1952) and in chicks by Jailer and Boas (1954) following ACTH injection, although no effect was noted in chicks by Conner (1959)_ or in quail by Flickinger (1959). Newcomer (1957) reported that ACTH injection produced leucocytosis and heterophilia in one- to two-week-old chicks but he could not find any consistent change in eosinophils, basophils or monocytes within 24 hours after the injection. Garren and Shaffner (1952, 1954, 1956) showed that cold (44°F.) exposure and muscular fatigue caused adrenal hypertrophy. As the population density of chickens increased, adrenal hypertrophy and adrenal cholesterol depletion were observed by Siegal (1959, 1960). The present studies were undertaken to determine if staining for the presence of neutral fats, assaying for adrenal cholesterol and abscorbic acid or performing a differential leucocyte count could be used as physiological indicators of stress in mature S. C. White Leghorn females.
INDICATORS OF STRESSOR AGENTS
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Depo-ACTH—A product of the Upjohn Company. 'Corticotropin ACTH—A product of Armour Pharmaceutical Company.
temperature control room (70-80°F.) for four days. ACTH and physiological saline were injected intramuscularly into the pectoralis muscle. The experiment was as follows: No. 1 Control—Non-injected No. 2 Physiological saline-injected control—0.5 c.c. twice daily, 8:00 a.m. and 5-7:00 p.m. for four days. No. 3 ACTH-injected—0.5 c.c, 5 I.U., twice daily, at 8:00 a.m. and 5-7:00 p.m. for four days. RESULTS
Experiment 1: The average adrenal ascorbic acid concentration (Table 2) was 215.3 and 167.1 mg. percent for layers and non-layers, respectively. These means were shown to be significantly different (P < 0.01). The right adrenals of nonlayers were 15.4 mg. heavier than those of layers. However, housing birds either on a conventional floor, in single cages or in colony cages did not significantly alter their adrenal ascorbic acid content, adrenal ascorbic acid concentration or adrenal weight (Table 1). Statistical analysis indicated that a single intravenous injection of ACTH (Table 3) did not cause any significant changes in adrenal weight, adrenal ascorbic acid content or adrenal ascorbic acid concentration. Experiment 2: In this experiment, handling procedures, cold exposure (35°F.) and intravenous ACTH injections did not significantly alter adrenal cholesterol or adrenal weight (Table 4). Exposing hens to 35°F. for 24 hours resulted in an increased eosinophilic percentage, from 1.7 to 6.8 percent. The data also showed that handling and ACTH caused an increase in the percentage of heterophils and/or a decrease in the percentage of lymphocytes. However, since there was no significant difference between the ACTH and handling
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lyzed by analysis of variance (Dixon and Massey, 1957) and Duncan's multiple range and multiple F tests (1955). In experiment 1, 35 layers and 11 nonlayers were randomly selected from hens housed either on a conventional floor or in single or colony cages. Eleven of the layers were injected intravenously with 10 I.U. of ACTH.6 The ACTH injected birds were killed approximately one hour and fifteen minutes after the injection while the other birds were killed immediately upon removal from their surroundings. The hens used in Experiment 2 were housed on a conventional floor throughout the entire production period. Twenty-four of the 33 birds kept under these conditions were removed and placed in individual cages for 24 hours. Eight of the 24 hens were exposed to a 35°F. temperature; and 16 hens were exposed to a temperature of 70-80° F., of which 8 hens were designated as handling controls and 8 hens were intravenously injected with 10 I.U. of ACTH7 before placing them in the cages. The birds used in Experiment 3 were housed in individual cages which were located in a temperature control room (7080°F.) for the entire production period. The birds were divided into five groups. Group 1 was designated as control and given feed and water ad libitum; Group 2 was not given feed for 40 hours prior to sacrificing; Group 3 was not given water for 24 hours prior to sacrificing; Group 4 was not given feed or water 15 hours prior to sacrificing; and Group 5 was exposed to cold (0°F.) and was not given feed or water 15 hours prior to sacrificing. In Experiment 4, 18 birds were placed in individual cages which were located in a
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J . H . WOLFORD AND R . K . RlNGER
TABLE 1.—Experiment J. Adrenal weight, ascorbic acid content and ascorbic acid concentration of layers and non-layers housed on a conventional floor, in single cages and colony cages Adrenal ascorbic acid Number of Body weight birds/group (kg.) L 1.83 1.90 1.87
NL 4 4 3
H 8 7 9
Conventional floor Single cages Colony cages Standard error of mean F-value
NL
Adrenal weight (mg.)
Content (meg.)
Concentration (meg. percent)
NL L NL L NL L 177.1 75.5 70.8 226.1 131.3 154.9 81.2 53.0 172.3 226.7 136.2 119.8 79.8 146.8 196.8 139.6 126.7 64.8 + 5.22 + 12.1 + 10.1 + 18.05 + 12.6 + 15.6 0.67 1.81 3.32 2.79 0.62 0.05
TABLE 2.—Experiment 1. Adrenal weight, ascorbic acid content and ascorbic acid concentration of layers vs. non-layers
Layers Non-layers Standard error of mean F-value
Number of birds/ group
Body wt. (kg.)
24 11
1.87 1.79
TABLE 3.—Experiment 1. Adrenal weight, ascorbic acid content and ascorbic acid concentration of laying hens following intravenous injection of A CTH
Adrenal ascorbic acid
+0.07 1.44
Adrenal wt. (mg.) 63.3 78.7 + 4.2 5.83*
Content (mcg.)
Adrenal ascorbic acid
Concentration (meg. percent)
138.3 135.3
215.3 167.1
+ 7.8 0.01
+8.3 10.81**
birds/ group Controls A C T H injected Standard error of mean F-value
* Significantly different at the P <0.05 level. " Significantly different a t the P < 0 . 0 1 level.
wt. (kg.)
Adrenal wt. (mg.)
1.87 2.12
63.3 65.1
+ 0.13 4.71*
Content Concentra(meg.) tion (meg. percent) 138.3 130.5 -8.0 0.09
±3.8 0.09
215.3 201.9 + 8.9 1.15
* Significantly different at the P <0.05 level.
treatments, it is suggested that the heterophil and lymphocyte change probably resulted only from the handling procedures used in this experiment. Experiment 3: Water deprivation, starvation or cold exposure (0°F.) produced no significant changes in adrenal choles-
terol or total adrenal weight (Table 5). However, body weight was significantly decreased (0.31, 0.19 and 0.29 kg.) when feed, water or feed and water were removed for 40 hours, 24 hours, and IS hours, respectively. Right adrenal weight increased 9.4 mg. and the eosinophils de-
TABLE 4.—Experiment 2. Eject of handling, cold (35°F.) and A CTH on adrenal activity • leucocyte counts TreatZ t
Treatment
No. of birds/ group
Body weight (kg.)
Adrenal cholesterol (mg.)' Total content
1 Control 2 Handling control 3 Cold 35° F . 4 A C T H 10 I.U. Standard Error of M e a n F value Significant (P <0.05)»
9(9) 8(7) 8(6) 8(6)
1.81 1.77 1.69 1.75 ±0.07 0.70
3.14 3.12 3.07 2.68 + 0.33 0.42
P «,100 adrenal
3.45 3.53 3.22 3.47 +0.25 0.31
Differential leucocyte count Adrenal weight (mg.) Total
Right
Left
Eosino- Hetero- Lympho- Monophils phils cytes cytes (Percent)
166.6 153.7 167.6 141.5 + 9.6 1.77
92.7 87.9 92.4 79.5 +5.9 1.10
73.8 65.7 75.2 62.0 +4.9 1.70
1.7 21.0 3.1 41.3 6.8 31.2 1.1 55.1 + 1.3 + 5.7 3.67* 6.53" 1-3 1-2 3-4 1-4
* Significantly different at the P < 0 . 0 5 level. ** Significantly different a t the P < 0 . 0 1 level. Cholesterol assay a n d calculations based on the right adrenal only. Numbers joined by a dash are significantly different ( P < 0 . 0 5 ) (Duncan multiple range and multiple F tests). ( ) N u m b e r within bracket represents number of birds laying.
1 2
72.7 50.8 57.4 40.4 +5.1 + 7.40*"' 1-2 1-4 3-4
4.7 4.5 3.9 3.1 2.4 0.5
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1 L = Layers. NL=Non-layers.
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TABLE 5.—Experiment 3. Effect of starving, water deprivation and cold (0°F.) on adrenal activity and differential leucocyte counts
No.
Treatment
1 Control 2 N o feed 3 N o water 4 N o feed or water 5 Cold 0 ° F . Standard error of mean F value Significant P < 0 . 0 5 2
No. of birds/ group
11(11) 12(11) 12(11) 12(11) 12(11)
Body weight (kg.)
Adrenal cholesterol (mg.)> Total P e , L 1 0 0 content adrenal 2.62 3.29 2.79 2.91 2.98 + 0.30 0.65
Total
Right
Eosinophils
Left
Hetero- L y m p h o - Monophils cytes cytes (Percent)
93.9 89.6 100.6 95.5 110.6 ±5.2 2.44
45.0 48.9 42.7 46.9 49.2 51.7 47.7 47.8 52.3 58.3 + 3.0 + 2.8 1.91 5.48** 1-5 2-5 3-5 4-5
17.0 75.8 3.6 3.5 41.6 50.2 4.1 3.3 24.5 70.0 2.3 2.8 22.4 69.7 4.3 2.9 33.8 61.5 3.8 0.5 + 3.5 + 3 . 4 ±0.07 + 0.5 4.77** 7.17** 7.09** 1.41 1-2 1-2 1-5 1-5 1-5 2-5 2-3 2-3 3-5 2-4 3-4 4-5 4-5
** Significantly different at the P <0.01 level. Cholesterol assay and calculations based on the right adrenal only. Numbers joined by a dash are significantly different (P <0.05) (Duncan multiple range and multiple F tests). ( ) Numbers in brackets represent the number of birds laying.
1 2
creased significantly (3.5 to 0.5 percent) from that of controls when hens were exposed to cold (0°F.) for 15 hours without feed and water. The heterophil percentage increased and/or lymphocyte percentage decreased significantly when feed was removed for 40 hours or when the hens were exposed to cold (0°F.) for 15 hours without feed and water. Experiment 4: ACTH injected intramuscularly twice daily for four consecutive days depleted adrenal cholesterol (Table 6). The cholesterol per 100 mg. adrenal was 3.83, 3.69 and 1.65 mg. respectively, for control, saline injected control and ACTH injected hens. Adrenal weight and leucocyte counts were not significantly al-
tered by either the injection of saline or ACTH. Experiment 4 was the only experiment in which a significant difference (P < 0.01) in neutral fat concentration was observed. The average scores were 3.0, 4.7 and 5.7 respectively, for ACTH-injected, salineinjected and control hens. The effect of ACTH on the neutral fat concentration of the interrenal tissue is shown in Figure 1. DISCUSSION The data obtained in Experiment 4 (Table 6) support the hypothesis that ACTH is. involved in interrenal activity. An intramuscular injection of ACTH twice daily for four consecutive days caused a
TABLE 6.—Experiment 4. Effect of A CTH on adrenal activity and differential leucocyte counts
No.
Treatment
No. of birds/ group
Body weight (kg.)
Adrenal cholesterol (mg.)'
Total
F
Differential leucocyte count Adrenal weight (mg.)
Zl°°
Total
Right
Left
3.83 3.69 1.65 0.40 9.50** 1-3 2-3
132.2 115.0 138.3 ±12.2 0.98
69.0 59.2 73.4 + 6.2 1.38
63.2 55.8 64.9 + 6.9 0.26
Eosino- Hetero- Lympho- Monophils phils cytes cytes
content adrenal 1 Control 2 Saline-inj. control 3 A C T H ini. Standard error of mean F value Significant P < 0 . 0 5 2
6(5) 6(6) 6(6)
1.73 1.75 1.63 ±0.06 1.04
2.81 2.15 1.23 + 0.31 + 6.37** 1-3 2-3
(Percent) 3.2 2.0 2.7 + 0.9 0.45
24.3 34.7 37.3 ±3.90 3.05
** Significantly different at the P<0.01 level. Cholesterol assay and calculations based on the right adrenal only. Numbers joined by a dash are significantly different (P<0.05) (Duncan multiple range and multiple F tests). ( ) Numbers in brackets represent the number of birds laying.
1 2
68.1 59.7 54.2 + 4.4 2.60
4.3 3.2 3.7 1.2 2.25
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1.85 1.31 1.50 1.54 1.34 1.66 1.56 1.26 1.70 1.75 ±0.06 ±0.16 3.97**! 1.54 1-2 1-3 1^1 2-5
Differential leucocyte count Adrenal weight (mg.)
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J . H . WOLFORD AND R. K . RlNGER
A. Photomicrograph (X 100) of an adrenal gland gland stained with Oil Red 0, from an ACTH-injected hen. Note the light areas within the dark staining interrenal tissue, indicating neutral fat depletion. B. Photomicrograph (X 100) of an adrenal gland stained with Oil Red O, from a control hen. C. Photomicrograph (X 430) of same field as in A. D. Photomicrograph (X 430) of same field as in B.
significant (P < 0.01) cholesterol depletion (2.18 mg. per 100 mg. adrenal). Hence, it appears that ACTH stimulates the adrenal to use cholesterol in the synthesis of the interrenal hormones (Sayers ct al., 1944, 1945, 1946). However, Experiments 1 and 2 (Tables 3 and 4) indicate that adrenal cholesterol and adrenal ascorbic acid depletion may be a function of time, dosage and/or route of administration since single intravenous injections of ACTH did not significantly alter cholesterol or ascorbic acid content. Although the avian
adrenal may have a greater degree of functional independence (Newcomer, 1959; Miller, 1961; Brown, 1961), the cholesterol depletion following ACTH injection suggests that an aclrenal-hypophyseal axis probably exists even though the other control centers may also initiate interrenal activity during rest and possibly during stress. A differential count showed that, in general, the percentage of lymphocytes decreased and the percentage of heterophils increased following exposure of the hens to
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FIGURE 1
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INDICATORS OF STRESSOR AGENTS
However these data are in disagreement with Perek and Eckstein (1959) who showed that ascorbic acid was depleted following a comparable ACTH injection. From the data obtained, it appears that differential leucocyte counts may possibly be used as stress indicators while adrenal weight, adrenal ascorbic acid and adrenal cholesterol appear to be unreliable indicators, at least in mature S. C. White Leghorn hens. However, the time allotted for the changes to occur may possibly explain the lack of cholesterol depletion from the adrenal following cold exposure, handling and feed and water deprivation. Therefore, it may be necessary to use other tests to measure stressor agents in laying hens. Such a test may be that of Brown (1961) in which he demonstrated that corticosterone is the principle free corticosteroid found in the plasma of six-week-old turkeys and that this steroid is elevated by ACTH, cold exposure and water deprivation. SUMMARY Experiments were conducted to evaluate adrenal weight, adrenal ascorbic acid, adrenal cholesterol and differential leucocyte counts as physiological indicators of stressor agents in mature S. C. White Leghorn hens. Single intravenous injections of ACTH did not significantly deplete either adrenal cholesterol or adrenal ascorbic acid; however, an intramuscular injection twice daily for four consecutive days caused a 67.9 percent reduction in the amount of cholesterol per 100 mg. adrenal. Adrenal cholesterol was not significantly altered by any other treatment employed in this study. In general, the percentage of lymphocytes decreased and/or the percentage of heterophils increased following the various treatments; although only handling, no
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stressor agents. However, handling (Table 4), feed deprivation for 40 hours (Table 5), intravenous injection of ACTH (Table 4), and exposure to cold (0°F.) for 15 hours without feed and water (Table 5), were the only factors which significantly altered the heterophil or lymphocyte percentage. The differences which occurred after an ACTH intravenous injection and exposure to cold may have resulted from the handling procedures involved. Thus, differential leucocyte counts may possibly be used as an indicator of interrenal activity; however, it is not possible from the experimental evidence presented in this study to determine which cells, heterophils or lymphocytes, were influenced by the treatments. In this study, adrenal weight appeared to be an unreliable reference of interrenal activity. In Experiment 1 (Table 2), the right adrenals of non-laying hens were significantly heavier than were those of laying hens. The right adrenals of birds exposed to cold and subjected to 15 hours without feed and water (Experiment 3, Table 5) were significantly heavier than the right adrenals of control birds, but total adrenal weights and left adrenal weights were not significantly altered. Adrenal weight did not appear to be influenced by any other treatment. The average weights of the adrenal glands of birds used in this study were: right, 66.0 mg.; left, 58.3 mg.; total, 124.3 mg. Thus, the right adrenals were heavier than the left adrenals. This evidence is in agreement with the results reported by Siegel (1960). No significant differences in adrenal ascorbic acid content and concentration were found between control laying hens and laying hens injected with ACTH (Table 3). This information is in agreement with the results reported by previous workers (Jailer and Boas, 1954; Zarrow and Zarrow, 1950; Zarrow and Baldini, 1952).
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J . H . WOLFORD AND R . K . RlNGER
ACKNOWLEDGMENT The authors gratefully acknowledge the technical assistance of Mrs. Marjorie J. Tetzlaff. REFERENCES Brown, K. I., D. J. Brown and R. K. Meyer, 1958. Effect of surgical trauma, ACTH and adrenal cortical hormones on electrolytes, water balance and gluconeogenesis in male chickens. Am. J. Physiol. 192 : 43-50. Brown, K. I., 1961. The validity of using plasma corticosterone as a measure of stress in the turkey. Proc. Soc. Exp. Biol. Med. 107: 538542. Conner, M. H., 1959. Effect of various hormone preparations and nutritional stresses in chicks. Poultry Sci. 38: 1340-1343. Dixon, W. J., and F. J. Massey, Jr., 1957. Introduction To Statistical Analysis. 2nd Ed. McGraw-Hill Company, Inc., New York. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 1 1 : 1-42. Elton, R. L., I. G. Zarrow and M. X. Zarrow,
1959. Depletion of adrenal ascorbic acid and cholesterol: A comparative study. Endocrinol. 65: 152-157. Flickinger, D. D., 1959. Adrenal responses of California quail subject to various physiologic stimuli. Proc. Soc. Exp. Biol. Med. 100: 23-25. Garren, H. W., and C. S. Shaffner, 1952. The response of young New Hampshire chickens to conditions of stress. Poultry Sci. 3 1 : 917. Garren, H. W., and C. S. Shaffner, 1954. Factors concerned in the response of young New Hampshires to muscular fatigue. Poultry Sci. 33: 1095-1104. Garren, H. W., and C. S. Shaffner, 1956. How the period of exposure to different stress stimuli affects the endocrine and lymphatic gland weight of young chickens. Poultry Sci. 35: 266-272. Howard, A. N., and B. J. Constable, 1958. The metabolism of adrenocorticotrophic hormone and ascorbic acid in the chick. Biochem. J. 69: 501-505. Jailer, J. W., and N. F. Boas, 1954. The inability of epinephrine or adrenocorticotropic hormone to deplete the ascorbic acid content of the chick adrenal. Endocrinol. 46: 314-318. Knobil, K., M. G. Hagney, E. J. Wilder and F. N. Briggs, 1954. Simplified method for determination of total adrenal cholesterol. Proc. Soc. Exp. Biol. Med. 87: 48-50. Lillie, R. D., 1954. Histopathologic Technic and Practical Histochemistry. Blakiston Co., New York, p. 303. Miller, R. A., 1961. Hypertrophic adrenals and their response to stress after lesions in the median eminence of totally hypophysectomized pigeons. Acta. Endocrinol. 37: 565-576. Newcomer, W. S., 1957. Blood cell changes following ACTH injection in the chick. Proc. Soc. Exp. Biol. Med. 96: 613-616. Newcomer, W. S., 1959. Effects of hypophysectomy on some functional aspects of the adrenal gland of the chicken. Endocrinol. 65: 133-135. Perek, M., and B. Eckstein, 1959. The adrenal ascorbic acid content of molting hens and the effect of ACTH on the adrenal ascorbic acid content of laying hens. Poultry Sci. 38: 996999. Roe, J. H., and C. A. Kuether, 1943. The determination of ascorbic acid in whole blood and urine through the 2,4-dinitrophenylhydrazine derivative of dehydroascorbic acid. J. Biol. Chem. 147: 399-407.
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feed for 40 hours and exposure to cold (0°F. for 15 hours without feed and water) significantly altered the lymphocyte and/ or the heterophil percentage. The average ascorbic acid concentration per 100 mg. adrenal was 215 meg. for layers and 167 meg. for non-layers. The average cholesterol concentration was 3.22 mg. per 100 mg. adrenal. The average total adrenal weight was 124 mg., with the right adrenal comprising 53.2 percent and the left comprising 46.8 percent of the total adrenal weight. Leucocyte percentage averaged 2, 3, 4, 28 and 63 percent respectively, for basophils, eosinophils, monocytes, heterophils and lymphocytes. From the data obtained, it appears that differential leucocyte counts may possibly be used as stress indicators while adrenal weight, adrenal ascorbic acid and adrenal cholesterol appear to be unreliable, at least in mature S. C. White Leghorn hens.
INDICATORS OF STRESSOR AGENTS
istics and adrenal function in White Leghorns confined at different floor space levels. Poultry Sci. 38: 893-898. Siegel, H. S., 1960. Effect of population density on the pituitary-adrenal cortical axis of cockerels. Poultry Sci. 39: 500-510. Siegel, H. S., and W. L. Beane, 1961. Time responses to single intramuscular doses of ACTH in chickens. Poultry Sci. 40: 216-219. Zarrow, M. X., and I. G. Zarrow, 1950. Ascorbic acid in the adrenal gland of the duck. Anat. Record, 108: 600-601. Zarrow, M. X., and J. F. Baldini, 1952. Failure of adrenocorticotropic and various stimuli to deplete the ascorbic acid content of the adrenal gland of the quail. Endocrinol. 50: 555-561.
Sources of Xanthophyll for Pigmentation in Broilers1 ROBERT G. RATCLIFF, ELBERT J. DAY, CLARENCE 0. GROGAN2 AND JAMES E. HILL Department of Poultry Husbandry, Mississippi State University, State College, Mississippi (Received for publication February 20, 1962)
A N INCREASED interest in pigmenta-**• tation has occurred as evidenced by the numerous recent papers concerning this subject (Anjaneylu et al., 1961; Farr et al., 1961; Mitchell et al, 1961; Morehouse, 1961; Wheeler and Turk, 1961; Tarver, 1961; Couch, 1961; Ratcliff et al., 1961; Hastings, 1961; and Williams, 1962). Research work has centered around (1) finding new sources of xanthophyll, (2) enhancement of utilization of xanthophyll, (3) determining relative pigmenting values of feedstuffs and (4) egg yolk pigmentation. Pigmentation problem with broilers appears to be less acute than formerly, except in certain areas. The greatest problem today is with eggs being produced for 'Mississippi Agricultural Experiment Station Journal Article no. 1005. 2 Agronomist, U. S. Department of Agriculture, A.R.S., and Mississippi State University.
breakout operations where uniform deep colored egg yolks are desired, but have been difficult to obtain through the use of natural ingredients which furnish xanthophyll. Dietary levels of IS to 25% of high quality alfalfa leaf meal were found to be necessary to produce intense yolk pigmentation, NEPA 5 to 8 color indexes (Couch, 1961). However, due to its low energy and high fiber content, performance might be adversely affected at these levels. Also, several commercial xanthophyll concentrates have been found to be effective, but have not been economical to use. Therefore, the answer might be in selecting feedstuffs that have high xanthophyll content. The present study was concerned with a comparison of strains of yellow corn, selected for high xanthophyll content, with regular yellow corn and clover meal versus alfalfa meal as sources of xanthophyll in broiler rations.
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Sayers, G., M. A. Sayers, H. L. Lewis and C. N. H. Long, 1944. Effect of adrenotropic hormone on ascorbic acid and cholesterol content of the adrenal. Proc. Soc. Exp. Biol. Med. 55: 238-239. Sayers, G., M. A. Sayers, T. Y. Liang and C. N. H. Long, 1945. The cholesterol and ascorbic acid content of the adrenal, liver, brain and plasma following hemorrhage. Endocrinol. 37; 96-110. Sayers, G., M. A. Sayers, T. Y. Liang and C. N. H. Long, 1946. The effect of pituitary adrenotrophic hormone on the cholesterol and ascorbic content of the adrenal of the rat and guinea pig. Endocrinol. 39: 1-9. Sayers, G., 1950. The adrenal and homeostasis. Physiol. Rev. 30: 241-320. Siegel, H. S., 1959. Egg production character-
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SUMMARY
A rapid method for the evaluation of the vitelline membrane strength of the intact egg yolk was reported and evaluated. This technique involves the placement of a 2 mm. capillary tube against the vitelline membrane, the creation of a given vacuum
in the capillary tube and measurement of the time required to rupture the membrane. The time required for rupture is plotted relative to a standard curve in order to equate it with time required at other vacuums or the vacuum required to rupture the vitelline membrane in one second. It was found that temperature of the egg yolk is a factor which affects the relative strength of the vitelline membrane as measured by the capillary-vacuum method. REFERENCES Haugh, R. R., 1933. A new method for determining the quality of an egg. U.S. Egg Poultry Mag. 39 : 27, 49. Moran, T., and H. P. Hale, 1936. Physics of the hen's egg. I. Membranes in the egg. J. Exp. Biol. 13: 3S-40. Moran, T., 1936. Physics of the hen's egg. II. The bursting strength of the vitelline membrane. J. Exp. Biol. 13 : 41^17. Munro, S. S., and G. Robertson, 1935. A method of measuring the strength of the yolk membrane. U.S. Egg Poultry Mag. 4 1 : 48, 50. Sharp, P. F., 1929. What one week may do to an egg. U.S. Egg Poultry Mag. 35: 14-17.
Adrenal Weight, Adrenal Ascorbic Acid, Adrenal Cholesterol and Differential Leucocyte Counts as Physiological Indicators of "Stressor" Agents in Laying Hens1,2 Department
JOHN H. WOLFORD AND ROBERT K. RINGER of Poultry Science, Michigan State University, East Lansing, Michigan (Received for publication February 19, 1962)
T is thought that the adrenal cortex assumes an important role in an animal's ability to overcome environmental influences. Therefore, assays capable of in-
I
1 Journal Article No. 2942, Michigan Agricultural Experiment Station. 2 Part of a doctoral dissertation submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree.
dicating adrenal cortical function following changes in management, feed, lighting schedules and weather, among other factors, would possibly be a step toward the elimination of production decreases. Research with rats and guinea pigs (reviewed by Sayers, 1950) has shown that depletion of adrenal ascorbic acid, cholesterol and lipid contents occurs following an
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degree of relative elasticity nor grams pressure required to rupture the vitelline membrane could be secured. The use of a 2 mm. capillary tube was found to be preferable since a large number of measurements could be made on each intact vitelline membrane before the yolk lost its physical identity. In addition to the comparable results acquired relative to the other physical methods described in this report, the general principle of the capillary-vacuum method for evaluating the strength of the vitelline membrane may also be used in securing small samples of the egg contents from a specific yolk area for microanalyses without disrupting the general physical nature of the egg yolk.
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J . H . WOLFORD AND R . K . RlNGEE EXPERIMENTAL PROCEDURE
S. C. \Vhite Leghorn females in production for more than 40 weeks and housed either on a conventional floor or in cages were used in this study. All birds were given feed and water ad libitum, except where noted in a particular experiment. All hens designated as layers had partially formed eggs in their oviducts or had several large follicles in their ovaries at the time of sacrifice for adrenal assay. The ovaries of those birds designated as non-layers were atresic and contained only undeveloped follicles. Adrenal ascorbic acid was determined by the method of Roe and Kuether (1943), and adrenal cholesterol was determined by the method of Knobil et al. (1954). In all cases only the right adrenal gland was utilized for either the ascorbic acid or cholesterol determination. The left adrenal gland was fixed in formalin and strained with Oil Red 0 to determine the amount of neutral fat present Lillie (1954).3 These stained sections were then scored on the basis of 1 and 2 = low; 3 and 4 = medium; and 5 and 6 = high neutral fat concentration. Colorimetric readings for cholesterol and ascorbic acid were taken on a "Universal Model 14" spectrometer. Prior to sacrificing by cervical dislocation, blood samples and smears4 were taken. The blood smears were stained with Wright's stain, and a differential leucocyte count was made. Following cervical dislocation, the birds were weighed and each adrenal gland, right and left,5 was immediately removed and weighed. The data obtained were statistically ana3
The left adrenal gland was not removed in the ascorbic acid experiment. 4 Blood samples and smears were not taken in the ascorbic acid experiment. 5 The left adrenal gland was not removed in the ascorbic acid experiment.
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injection of pituitary adrenotropic hormone and following stressful conditions such as excessive muscular work or a non-fatal hemorrhage. Reports involving "stressor" agents and their influence on the avian adrenal have been more inconsistent than the reported influence on the mammalian adrenal. Howard and Constable (1958) and Siegel and Beane (1961) produced adrenal cholesterol depletion in chickens following ACTH injection; however, Elton et al. (1959) reported that adrenal cholesterol remained unchanged following ACTH and cold (0-5°C.) treatment. Perek and Eckstein (1959) were able to elicit adrenal ascorbic acid depletion in laying hens following ACTH injection. Adrenal hypertrophy, another measure of interrenal activity has been shown in male chickens by Brown et al. (1958), in quail by Zarrow and Baldini (1952) and in chicks by Jailer and Boas (1954) following ACTH injection, although no effect was noted in chicks by Conner (1959)_ or in quail by Flickinger (1959). Newcomer (1957) reported that ACTH injection produced leucocytosis and heterophilia in one- to two-week-old chicks but he could not find any consistent change in eosinophils, basophils or monocytes within 24 hours after the injection. Garren and Shaffner (1952, 1954, 1956) showed that cold (44°F.) exposure and muscular fatigue caused adrenal hypertrophy. As the population density of chickens increased, adrenal hypertrophy and adrenal cholesterol depletion were observed by Siegal (1959, 1960). The present studies were undertaken to determine if staining for the presence of neutral fats, assaying for adrenal cholesterol and abscorbic acid or performing a differential leucocyte count could be used as physiological indicators of stress in mature S. C. White Leghorn females.
INDICATORS OF STRESSOR AGENTS
6
Depo-ACTH—A product of the Upjohn Company. 'Corticotropin ACTH—A product of Armour Pharmaceutical Company.
temperature control room (70-80°F.) for four days. ACTH and physiological saline were injected intramuscularly into the pectoralis muscle. The experiment was as follows: No. 1 Control—Non-injected No. 2 Physiological saline-injected control—0.5 c.c. twice daily, 8:00 a.m. and 5-7:00 p.m. for four days. No. 3 ACTH-injected—0.5 c.c, 5 I.U., twice daily, at 8:00 a.m. and 5-7:00 p.m. for four days. RESULTS
Experiment 1: The average adrenal ascorbic acid concentration (Table 2) was 215.3 and 167.1 mg. percent for layers and non-layers, respectively. These means were shown to be significantly different (P < 0.01). The right adrenals of nonlayers were 15.4 mg. heavier than those of layers. However, housing birds either on a conventional floor, in single cages or in colony cages did not significantly alter their adrenal ascorbic acid content, adrenal ascorbic acid concentration or adrenal weight (Table 1). Statistical analysis indicated that a single intravenous injection of ACTH (Table 3) did not cause any significant changes in adrenal weight, adrenal ascorbic acid content or adrenal ascorbic acid concentration. Experiment 2: In this experiment, handling procedures, cold exposure (35°F.) and intravenous ACTH injections did not significantly alter adrenal cholesterol or adrenal weight (Table 4). Exposing hens to 35°F. for 24 hours resulted in an increased eosinophilic percentage, from 1.7 to 6.8 percent. The data also showed that handling and ACTH caused an increase in the percentage of heterophils and/or a decrease in the percentage of lymphocytes. However, since there was no significant difference between the ACTH and handling
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lyzed by analysis of variance (Dixon and Massey, 1957) and Duncan's multiple range and multiple F tests (1955). In experiment 1, 35 layers and 11 nonlayers were randomly selected from hens housed either on a conventional floor or in single or colony cages. Eleven of the layers were injected intravenously with 10 I.U. of ACTH.6 The ACTH injected birds were killed approximately one hour and fifteen minutes after the injection while the other birds were killed immediately upon removal from their surroundings. The hens used in Experiment 2 were housed on a conventional floor throughout the entire production period. Twenty-four of the 33 birds kept under these conditions were removed and placed in individual cages for 24 hours. Eight of the 24 hens were exposed to a 35°F. temperature; and 16 hens were exposed to a temperature of 70-80° F., of which 8 hens were designated as handling controls and 8 hens were intravenously injected with 10 I.U. of ACTH7 before placing them in the cages. The birds used in Experiment 3 were housed in individual cages which were located in a temperature control room (7080°F.) for the entire production period. The birds were divided into five groups. Group 1 was designated as control and given feed and water ad libitum; Group 2 was not given feed for 40 hours prior to sacrificing; Group 3 was not given water for 24 hours prior to sacrificing; Group 4 was not given feed or water 15 hours prior to sacrificing; and Group 5 was exposed to cold (0°F.) and was not given feed or water 15 hours prior to sacrificing. In Experiment 4, 18 birds were placed in individual cages which were located in a
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J . H . WOLFORD AND R . K . RlNGER
TABLE 1.—Experiment J. Adrenal weight, ascorbic acid content and ascorbic acid concentration of layers and non-layers housed on a conventional floor, in single cages and colony cages Adrenal ascorbic acid Number of Body weight birds/group (kg.) L 1.83 1.90 1.87
NL 4 4 3
H 8 7 9
Conventional floor Single cages Colony cages Standard error of mean F-value
NL
Adrenal weight (mg.)
Content (meg.)
Concentration (meg. percent)
NL L NL L NL L 177.1 75.5 70.8 226.1 131.3 154.9 81.2 53.0 172.3 226.7 136.2 119.8 79.8 146.8 196.8 139.6 126.7 64.8 + 5.22 + 12.1 + 10.1 + 18.05 + 12.6 + 15.6 0.67 1.81 3.32 2.79 0.62 0.05
TABLE 2.—Experiment 1. Adrenal weight, ascorbic acid content and ascorbic acid concentration of layers vs. non-layers
Layers Non-layers Standard error of mean F-value
Number of birds/ group
Body wt. (kg.)
24 11
1.87 1.79
TABLE 3.—Experiment 1. Adrenal weight, ascorbic acid content and ascorbic acid concentration of laying hens following intravenous injection of A CTH
Adrenal ascorbic acid
+0.07 1.44
Adrenal wt. (mg.) 63.3 78.7 + 4.2 5.83*
Content (mcg.)
Adrenal ascorbic acid
Concentration (meg. percent)
138.3 135.3
215.3 167.1
+ 7.8 0.01
+8.3 10.81**
birds/ group Controls A C T H injected Standard error of mean F-value
* Significantly different at the P <0.05 level. " Significantly different a t the P < 0 . 0 1 level.
wt. (kg.)
Adrenal wt. (mg.)
1.87 2.12
63.3 65.1
+ 0.13 4.71*
Content Concentra(meg.) tion (meg. percent) 138.3 130.5 -8.0 0.09
±3.8 0.09
215.3 201.9 + 8.9 1.15
* Significantly different at the P <0.05 level.
treatments, it is suggested that the heterophil and lymphocyte change probably resulted only from the handling procedures used in this experiment. Experiment 3: Water deprivation, starvation or cold exposure (0°F.) produced no significant changes in adrenal choles-
terol or total adrenal weight (Table 5). However, body weight was significantly decreased (0.31, 0.19 and 0.29 kg.) when feed, water or feed and water were removed for 40 hours, 24 hours, and IS hours, respectively. Right adrenal weight increased 9.4 mg. and the eosinophils de-
TABLE 4.—Experiment 2. Eject of handling, cold (35°F.) and A CTH on adrenal activity • leucocyte counts TreatZ t
Treatment
No. of birds/ group
Body weight (kg.)
Adrenal cholesterol (mg.)' Total content
1 Control 2 Handling control 3 Cold 35° F . 4 A C T H 10 I.U. Standard Error of M e a n F value Significant (P <0.05)»
9(9) 8(7) 8(6) 8(6)
1.81 1.77 1.69 1.75 ±0.07 0.70
3.14 3.12 3.07 2.68 + 0.33 0.42
P «,100 adrenal
3.45 3.53 3.22 3.47 +0.25 0.31
Differential leucocyte count Adrenal weight (mg.) Total
Right
Left
Eosino- Hetero- Lympho- Monophils phils cytes cytes (Percent)
166.6 153.7 167.6 141.5 + 9.6 1.77
92.7 87.9 92.4 79.5 +5.9 1.10
73.8 65.7 75.2 62.0 +4.9 1.70
1.7 21.0 3.1 41.3 6.8 31.2 1.1 55.1 + 1.3 + 5.7 3.67* 6.53" 1-3 1-2 3-4 1-4
* Significantly different at the P < 0 . 0 5 level. ** Significantly different a t the P < 0 . 0 1 level. Cholesterol assay a n d calculations based on the right adrenal only. Numbers joined by a dash are significantly different ( P < 0 . 0 5 ) (Duncan multiple range and multiple F tests). ( ) N u m b e r within bracket represents number of birds laying.
1 2
72.7 50.8 57.4 40.4 +5.1 + 7.40*"' 1-2 1-4 3-4
4.7 4.5 3.9 3.1 2.4 0.5
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1 L = Layers. NL=Non-layers.
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INDICATORS OF STRESSOR AGENTS
TABLE 5.—Experiment 3. Effect of starving, water deprivation and cold (0°F.) on adrenal activity and differential leucocyte counts
No.
Treatment
1 Control 2 N o feed 3 N o water 4 N o feed or water 5 Cold 0 ° F . Standard error of mean F value Significant P < 0 . 0 5 2
No. of birds/ group
11(11) 12(11) 12(11) 12(11) 12(11)
Body weight (kg.)
Adrenal cholesterol (mg.)> Total P e , L 1 0 0 content adrenal 2.62 3.29 2.79 2.91 2.98 + 0.30 0.65
Total
Right
Eosinophils
Left
Hetero- L y m p h o - Monophils cytes cytes (Percent)
93.9 89.6 100.6 95.5 110.6 ±5.2 2.44
45.0 48.9 42.7 46.9 49.2 51.7 47.7 47.8 52.3 58.3 + 3.0 + 2.8 1.91 5.48** 1-5 2-5 3-5 4-5
17.0 75.8 3.6 3.5 41.6 50.2 4.1 3.3 24.5 70.0 2.3 2.8 22.4 69.7 4.3 2.9 33.8 61.5 3.8 0.5 + 3.5 + 3 . 4 ±0.07 + 0.5 4.77** 7.17** 7.09** 1.41 1-2 1-2 1-5 1-5 1-5 2-5 2-3 2-3 3-5 2-4 3-4 4-5 4-5
** Significantly different at the P <0.01 level. Cholesterol assay and calculations based on the right adrenal only. Numbers joined by a dash are significantly different (P <0.05) (Duncan multiple range and multiple F tests). ( ) Numbers in brackets represent the number of birds laying.
1 2
creased significantly (3.5 to 0.5 percent) from that of controls when hens were exposed to cold (0°F.) for 15 hours without feed and water. The heterophil percentage increased and/or lymphocyte percentage decreased significantly when feed was removed for 40 hours or when the hens were exposed to cold (0°F.) for 15 hours without feed and water. Experiment 4: ACTH injected intramuscularly twice daily for four consecutive days depleted adrenal cholesterol (Table 6). The cholesterol per 100 mg. adrenal was 3.83, 3.69 and 1.65 mg. respectively, for control, saline injected control and ACTH injected hens. Adrenal weight and leucocyte counts were not significantly al-
tered by either the injection of saline or ACTH. Experiment 4 was the only experiment in which a significant difference (P < 0.01) in neutral fat concentration was observed. The average scores were 3.0, 4.7 and 5.7 respectively, for ACTH-injected, salineinjected and control hens. The effect of ACTH on the neutral fat concentration of the interrenal tissue is shown in Figure 1. DISCUSSION The data obtained in Experiment 4 (Table 6) support the hypothesis that ACTH is. involved in interrenal activity. An intramuscular injection of ACTH twice daily for four consecutive days caused a
TABLE 6.—Experiment 4. Effect of A CTH on adrenal activity and differential leucocyte counts
No.
Treatment
No. of birds/ group
Body weight (kg.)
Adrenal cholesterol (mg.)'
Total
F
Differential leucocyte count Adrenal weight (mg.)
Zl°°
Total
Right
Left
3.83 3.69 1.65 0.40 9.50** 1-3 2-3
132.2 115.0 138.3 ±12.2 0.98
69.0 59.2 73.4 + 6.2 1.38
63.2 55.8 64.9 + 6.9 0.26
Eosino- Hetero- Lympho- Monophils phils cytes cytes
content adrenal 1 Control 2 Saline-inj. control 3 A C T H ini. Standard error of mean F value Significant P < 0 . 0 5 2
6(5) 6(6) 6(6)
1.73 1.75 1.63 ±0.06 1.04
2.81 2.15 1.23 + 0.31 + 6.37** 1-3 2-3
(Percent) 3.2 2.0 2.7 + 0.9 0.45
24.3 34.7 37.3 ±3.90 3.05
** Significantly different at the P<0.01 level. Cholesterol assay and calculations based on the right adrenal only. Numbers joined by a dash are significantly different (P<0.05) (Duncan multiple range and multiple F tests). ( ) Numbers in brackets represent the number of birds laying.
1 2
68.1 59.7 54.2 + 4.4 2.60
4.3 3.2 3.7 1.2 2.25
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1.85 1.31 1.50 1.54 1.34 1.66 1.56 1.26 1.70 1.75 ±0.06 ±0.16 3.97**! 1.54 1-2 1-3 1^1 2-5
Differential leucocyte count Adrenal weight (mg.)
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J . H . WOLFORD AND R. K . RlNGER
A. Photomicrograph (X 100) of an adrenal gland gland stained with Oil Red 0, from an ACTH-injected hen. Note the light areas within the dark staining interrenal tissue, indicating neutral fat depletion. B. Photomicrograph (X 100) of an adrenal gland stained with Oil Red O, from a control hen. C. Photomicrograph (X 430) of same field as in A. D. Photomicrograph (X 430) of same field as in B.
significant (P < 0.01) cholesterol depletion (2.18 mg. per 100 mg. adrenal). Hence, it appears that ACTH stimulates the adrenal to use cholesterol in the synthesis of the interrenal hormones (Sayers ct al., 1944, 1945, 1946). However, Experiments 1 and 2 (Tables 3 and 4) indicate that adrenal cholesterol and adrenal ascorbic acid depletion may be a function of time, dosage and/or route of administration since single intravenous injections of ACTH did not significantly alter cholesterol or ascorbic acid content. Although the avian
adrenal may have a greater degree of functional independence (Newcomer, 1959; Miller, 1961; Brown, 1961), the cholesterol depletion following ACTH injection suggests that an aclrenal-hypophyseal axis probably exists even though the other control centers may also initiate interrenal activity during rest and possibly during stress. A differential count showed that, in general, the percentage of lymphocytes decreased and the percentage of heterophils increased following exposure of the hens to
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FIGURE 1
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INDICATORS OF STRESSOR AGENTS
However these data are in disagreement with Perek and Eckstein (1959) who showed that ascorbic acid was depleted following a comparable ACTH injection. From the data obtained, it appears that differential leucocyte counts may possibly be used as stress indicators while adrenal weight, adrenal ascorbic acid and adrenal cholesterol appear to be unreliable indicators, at least in mature S. C. White Leghorn hens. However, the time allotted for the changes to occur may possibly explain the lack of cholesterol depletion from the adrenal following cold exposure, handling and feed and water deprivation. Therefore, it may be necessary to use other tests to measure stressor agents in laying hens. Such a test may be that of Brown (1961) in which he demonstrated that corticosterone is the principle free corticosteroid found in the plasma of six-week-old turkeys and that this steroid is elevated by ACTH, cold exposure and water deprivation. SUMMARY Experiments were conducted to evaluate adrenal weight, adrenal ascorbic acid, adrenal cholesterol and differential leucocyte counts as physiological indicators of stressor agents in mature S. C. White Leghorn hens. Single intravenous injections of ACTH did not significantly deplete either adrenal cholesterol or adrenal ascorbic acid; however, an intramuscular injection twice daily for four consecutive days caused a 67.9 percent reduction in the amount of cholesterol per 100 mg. adrenal. Adrenal cholesterol was not significantly altered by any other treatment employed in this study. In general, the percentage of lymphocytes decreased and/or the percentage of heterophils increased following the various treatments; although only handling, no
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stressor agents. However, handling (Table 4), feed deprivation for 40 hours (Table 5), intravenous injection of ACTH (Table 4), and exposure to cold (0°F.) for 15 hours without feed and water (Table 5), were the only factors which significantly altered the heterophil or lymphocyte percentage. The differences which occurred after an ACTH intravenous injection and exposure to cold may have resulted from the handling procedures involved. Thus, differential leucocyte counts may possibly be used as an indicator of interrenal activity; however, it is not possible from the experimental evidence presented in this study to determine which cells, heterophils or lymphocytes, were influenced by the treatments. In this study, adrenal weight appeared to be an unreliable reference of interrenal activity. In Experiment 1 (Table 2), the right adrenals of non-laying hens were significantly heavier than were those of laying hens. The right adrenals of birds exposed to cold and subjected to 15 hours without feed and water (Experiment 3, Table 5) were significantly heavier than the right adrenals of control birds, but total adrenal weights and left adrenal weights were not significantly altered. Adrenal weight did not appear to be influenced by any other treatment. The average weights of the adrenal glands of birds used in this study were: right, 66.0 mg.; left, 58.3 mg.; total, 124.3 mg. Thus, the right adrenals were heavier than the left adrenals. This evidence is in agreement with the results reported by Siegel (1960). No significant differences in adrenal ascorbic acid content and concentration were found between control laying hens and laying hens injected with ACTH (Table 3). This information is in agreement with the results reported by previous workers (Jailer and Boas, 1954; Zarrow and Zarrow, 1950; Zarrow and Baldini, 1952).
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J . H . WOLFORD AND R . K . RlNGER
ACKNOWLEDGMENT The authors gratefully acknowledge the technical assistance of Mrs. Marjorie J. Tetzlaff. REFERENCES Brown, K. I., D. J. Brown and R. K. Meyer, 1958. Effect of surgical trauma, ACTH and adrenal cortical hormones on electrolytes, water balance and gluconeogenesis in male chickens. Am. J. Physiol. 192 : 43-50. Brown, K. I., 1961. The validity of using plasma corticosterone as a measure of stress in the turkey. Proc. Soc. Exp. Biol. Med. 107: 538542. Conner, M. H., 1959. Effect of various hormone preparations and nutritional stresses in chicks. Poultry Sci. 38: 1340-1343. Dixon, W. J., and F. J. Massey, Jr., 1957. Introduction To Statistical Analysis. 2nd Ed. McGraw-Hill Company, Inc., New York. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 1 1 : 1-42. Elton, R. L., I. G. Zarrow and M. X. Zarrow,
1959. Depletion of adrenal ascorbic acid and cholesterol: A comparative study. Endocrinol. 65: 152-157. Flickinger, D. D., 1959. Adrenal responses of California quail subject to various physiologic stimuli. Proc. Soc. Exp. Biol. Med. 100: 23-25. Garren, H. W., and C. S. Shaffner, 1952. The response of young New Hampshire chickens to conditions of stress. Poultry Sci. 3 1 : 917. Garren, H. W., and C. S. Shaffner, 1954. Factors concerned in the response of young New Hampshires to muscular fatigue. Poultry Sci. 33: 1095-1104. Garren, H. W., and C. S. Shaffner, 1956. How the period of exposure to different stress stimuli affects the endocrine and lymphatic gland weight of young chickens. Poultry Sci. 35: 266-272. Howard, A. N., and B. J. Constable, 1958. The metabolism of adrenocorticotrophic hormone and ascorbic acid in the chick. Biochem. J. 69: 501-505. Jailer, J. W., and N. F. Boas, 1954. The inability of epinephrine or adrenocorticotropic hormone to deplete the ascorbic acid content of the chick adrenal. Endocrinol. 46: 314-318. Knobil, K., M. G. Hagney, E. J. Wilder and F. N. Briggs, 1954. Simplified method for determination of total adrenal cholesterol. Proc. Soc. Exp. Biol. Med. 87: 48-50. Lillie, R. D., 1954. Histopathologic Technic and Practical Histochemistry. Blakiston Co., New York, p. 303. Miller, R. A., 1961. Hypertrophic adrenals and their response to stress after lesions in the median eminence of totally hypophysectomized pigeons. Acta. Endocrinol. 37: 565-576. Newcomer, W. S., 1957. Blood cell changes following ACTH injection in the chick. Proc. Soc. Exp. Biol. Med. 96: 613-616. Newcomer, W. S., 1959. Effects of hypophysectomy on some functional aspects of the adrenal gland of the chicken. Endocrinol. 65: 133-135. Perek, M., and B. Eckstein, 1959. The adrenal ascorbic acid content of molting hens and the effect of ACTH on the adrenal ascorbic acid content of laying hens. Poultry Sci. 38: 996999. Roe, J. H., and C. A. Kuether, 1943. The determination of ascorbic acid in whole blood and urine through the 2,4-dinitrophenylhydrazine derivative of dehydroascorbic acid. J. Biol. Chem. 147: 399-407.
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feed for 40 hours and exposure to cold (0°F. for 15 hours without feed and water) significantly altered the lymphocyte and/ or the heterophil percentage. The average ascorbic acid concentration per 100 mg. adrenal was 215 meg. for layers and 167 meg. for non-layers. The average cholesterol concentration was 3.22 mg. per 100 mg. adrenal. The average total adrenal weight was 124 mg., with the right adrenal comprising 53.2 percent and the left comprising 46.8 percent of the total adrenal weight. Leucocyte percentage averaged 2, 3, 4, 28 and 63 percent respectively, for basophils, eosinophils, monocytes, heterophils and lymphocytes. From the data obtained, it appears that differential leucocyte counts may possibly be used as stress indicators while adrenal weight, adrenal ascorbic acid and adrenal cholesterol appear to be unreliable, at least in mature S. C. White Leghorn hens.
INDICATORS OF STRESSOR AGENTS
istics and adrenal function in White Leghorns confined at different floor space levels. Poultry Sci. 38: 893-898. Siegel, H. S., 1960. Effect of population density on the pituitary-adrenal cortical axis of cockerels. Poultry Sci. 39: 500-510. Siegel, H. S., and W. L. Beane, 1961. Time responses to single intramuscular doses of ACTH in chickens. Poultry Sci. 40: 216-219. Zarrow, M. X., and I. G. Zarrow, 1950. Ascorbic acid in the adrenal gland of the duck. Anat. Record, 108: 600-601. Zarrow, M. X., and J. F. Baldini, 1952. Failure of adrenocorticotropic and various stimuli to deplete the ascorbic acid content of the adrenal gland of the quail. Endocrinol. 50: 555-561.
Sources of Xanthophyll for Pigmentation in Broilers1 ROBERT G. RATCLIFF, ELBERT J. DAY, CLARENCE 0. GROGAN2 AND JAMES E. HILL Department of Poultry Husbandry, Mississippi State University, State College, Mississippi (Received for publication February 20, 1962)
A N INCREASED interest in pigmenta-**• tation has occurred as evidenced by the numerous recent papers concerning this subject (Anjaneylu et al., 1961; Farr et al., 1961; Mitchell et al, 1961; Morehouse, 1961; Wheeler and Turk, 1961; Tarver, 1961; Couch, 1961; Ratcliff et al., 1961; Hastings, 1961; and Williams, 1962). Research work has centered around (1) finding new sources of xanthophyll, (2) enhancement of utilization of xanthophyll, (3) determining relative pigmenting values of feedstuffs and (4) egg yolk pigmentation. Pigmentation problem with broilers appears to be less acute than formerly, except in certain areas. The greatest problem today is with eggs being produced for 'Mississippi Agricultural Experiment Station Journal Article no. 1005. 2 Agronomist, U. S. Department of Agriculture, A.R.S., and Mississippi State University.
breakout operations where uniform deep colored egg yolks are desired, but have been difficult to obtain through the use of natural ingredients which furnish xanthophyll. Dietary levels of IS to 25% of high quality alfalfa leaf meal were found to be necessary to produce intense yolk pigmentation, NEPA 5 to 8 color indexes (Couch, 1961). However, due to its low energy and high fiber content, performance might be adversely affected at these levels. Also, several commercial xanthophyll concentrates have been found to be effective, but have not been economical to use. Therefore, the answer might be in selecting feedstuffs that have high xanthophyll content. The present study was concerned with a comparison of strains of yellow corn, selected for high xanthophyll content, with regular yellow corn and clover meal versus alfalfa meal as sources of xanthophyll in broiler rations.
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Sayers, G., M. A. Sayers, H. L. Lewis and C. N. H. Long, 1944. Effect of adrenotropic hormone on ascorbic acid and cholesterol content of the adrenal. Proc. Soc. Exp. Biol. Med. 55: 238-239. Sayers, G., M. A. Sayers, T. Y. Liang and C. N. H. Long, 1945. The cholesterol and ascorbic acid content of the adrenal, liver, brain and plasma following hemorrhage. Endocrinol. 37; 96-110. Sayers, G., M. A. Sayers, T. Y. Liang and C. N. H. Long, 1946. The effect of pituitary adrenotrophic hormone on the cholesterol and ascorbic content of the adrenal of the rat and guinea pig. Endocrinol. 39: 1-9. Sayers, G., 1950. The adrenal and homeostasis. Physiol. Rev. 30: 241-320. Siegel, H. S., 1959. Egg production character-
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