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Effects of Different Environmental Temperatures on Urinary Estrogens of Maturing Fowl1
66
C. L. QUARLES, R. F. GENTRY AND G. 0 . BRESSLER
REFERENCES Andersen, A. A., 1958. New sampler for the collection sizing and conservation of viable airborne particles. J. Bact. 76: 471-484. Andersen, A. A., 1965. A sampler for respiratory health hazard assessment. Presented at the AIHA Conferences, May 6, 1965 at Houston, Texas. Andersen, A. A., and M. R. Andersen, 1961. A monitor for airborne bacteria. Applied Microbiol. 10: 181-184. Bressler, G. 0., 1961. Solar and windowless laying houses compared. Pennsylvania Agricultural Experiment Station, Science for the Farmer, Vol. IX, No. 2:3. Bressler, G. O., and A. J. G. Maw, 1966. High density and environmental control. Key to efficient egg production. Proceedings Thirteenth World's Poultry Congress, pages 468-473. Bressler, G. O., H. D. Bartlett and C. L. Quarles,
1967. Unpublished data at the Pennsylvania Experimental Station Farms. Chute, H. L., and M. Gershman, 1961. A new approach to hatchery sanitation. Poultry Sci. 40: 568-571. Chute, H. L., M. Gershman, R. M. Sherman and D. C. O'Meara, 1963. A program for sanitation for Maine chick hatcheries. Report Number 100, Maine Agricultural Experiment Station. Gentry, R. F., M. Mitrovic and G. R. Bubash, 1962. Application of Andersen sampler in hatchery sanitation. Poultry Sci. 41: 794-804. Gentry, R. F., 1968. Pennsylvania State University. Personal communications. Gentry, R. F., and C. L. Quarles, 1967. The measurement of bacterial contamination on egg shells. Poultry Sci. 46: 1263. Graves, R. C , and D. W. MacLaury, 1962. The effects of temperature, vapor pressure and absolute humidity on bacterial contamination of shell eggs. Poultry Sci. 41: 1219-1225. McNally, E. H., 1954. The relation of temperature and humidity to infection of shell eggs by bacteria. Section Papers of the 10th World Poultry Congress, page 107. Snedecor, G. W., 1956. Statistical Methods. The Iowa State University Press, Ames, Iowa. Williams, J. E., and A. D. Whittemore, 1967. A method for studying microbial penetration through the outer structures of the avian egg. Avian Diseases, 11: 467.
Effects of Different Environmental Temperatures on Urinary Estrogens of Maturing Fowl1 FRANK Y. TANG, TILL M. HUSTON AND HARDY M. EDWARDS, JR. Department of Poultry Science, University of Georgia, Athens, Georgia 30601 (Received for publication August 5, 1969)
T
HE principal estrogens of the domestic fowl are estrone, 17/3-estradiol estriol and 16-epiestriol (Mulay et al., 1968) Previous data (Mathur et al., 1966) has emphasized the significant differences in the ratios of urinary estrogens in the laying hen. The principal estrogens of the
1 University of Georgia, College of Agriculture, Experimental Stations Journal Series Paper 571, College Station, Athens, Georgia 30601.
domestic fowl, appearing in the urine in decreasing amounts, are estrone, 17^-estradiol, 16-espiestriol and estriol. It is not known, however, if there occurs any change in such ratios during the process of sexual maturation in the domestic fowl. In the prepubertal bull calf, for example, it has been shown (Linder, 1959; Linder and Mann, 1960) that androstenedion predominates while in adulthood, the re-
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5. Bacterial counts on the eggs produced in litter floor houses were 20 to 30 times greater than on eggs produced in wire floor houses. 6. The presence of coliform bacteria in embryos and chicks was related to their concentration in the air of the poultry house.
TEMPERATURE EFFECTS ON ESTROGEN
MATERIALS AND METHODS
Single Comb White Leghorn pullets (Kimber 137) raised in an uncontrolled ambient temperature were colostomized at 17 weeks of age. Subsequently, operated birds were separated at random into environmental temperature regimes of 19°C. and 30°C. with a diurnal cycle of 15-23°C. and 25-34°C. respectively. Four to five days following the operation, each colostomized bird was fitted with a self-retaining tygon cannulae inserted into the exteriorized rectum to prevent occlusion. Forty-eight hour urine collections were made from each modified bird at 24, 26, 28, and 30 weeks of age. To minimize bacterial action during the collection period, the collection apparatus was dusted with powdered penicillin G and dihydrostreptomycin sulfate. Urine aliquots were transferred every eight hours to individual quart-size containers, frozen immediately at — 15°C. pending chemical analysis. Estrogen Analysis. Analytical grade reagents were distilled in all-glass apparatus before use. To test for impurities, quantities of solvent corresponding in volume to the amount used during the procedure were evaporated to dryness and the residue injected with acetone into the gas chromatograph (Wotiz and Chattoraj, 1964). The same operation was performed
with petroleum ether (30-60°C. b.p.) after shaking with a 5:1 mixture of acetic anhydride :pyridine. In any perceptible peaks appeared corresponding in retention time to that of the estrogen acetates, the solvent was redistilled and the test procedure repeated. Estrogen analyses were made according to the procedure of Brown (1955) and Wotiz and Chattoraj (1964) with the following modifications. Twenty-four hour urine aliquots were filtered with suction through spun glass wool. Following acid hydrolysis, care was taken to wash the boiling chips thoroughly with diethyl ether in order to prevent sizeable losses due to absorption on the stones. The dried phenolic extract was subjected to thin layer chromatography along with reference standards in benzenejethyl acetate ( l : l v / v ) in a semi-saturated chamber-lining only the two side walls of the developing tank. After staining the reference standards with dichlorofluorescein and visualization under ultraviolet light, the corresponding areas of the urine sample were eluted individually with ethanol. Following acetylation, derivatives were chromatographed on thin layer with reference estrogen acetates, using benzene: isopropyl either (9:1) in an incompletely saturated chamber (Varon et al., 1967). Rf values for EiA=.36, E 2 A=.56, E3A = .30, and E
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verse case exists with testosterone predominating. Recently it has been shown by Subhas and Huston (1967) that environmental temperatures may alter the normal balance of gonadotrophic hormones in the maturing female fowl. The development of a sensitive and specific method of assay applicable to fowl urinary estrogens makes it possible to examine in greater detail the estrogen picture in the maturing domestic fowl and the influence of environmental temperature on estrogen.
67
68
F. Y. T A N G , T. M . H U S T O N AND H. M .
Recovery studies were made b y adding a measured quantity of tritium ( 3 H) labelled estrogen dissolved in absolute ethanol representing approximately 70,000 c.m.p. to a 24 hour sample or urine before acid hydrolysis. Aliquots were taken at various steps during the extraction procedure as a check on recoveries u p to gas chromatography. T h e aliquots were evaporated down to dryness in a scintillation vial. Then. 10 ml. scintillation fluid which contained 5 grams of 2,5-diphenyloxazole and l,4-bis-2(5-phenyloxazolyl)-benzene per liter of toluene was added, and the entire vial placed in a Packard Tricarb liquid scintillation spectrometer and counted for 5 minutes. T h e efficiency for tritium on this instrument was 29.0%. Corrections were made for background and quenching.
Collections of effluent from the gas chromatograph were accomplished b y means of a variable stream splitter beyond the column so t h a t a portion of t h e sample could be vented to the outside for collection. As the desired peak appeared, the capillary end of a glass collection tube containing a wad of glass wool soaked in acetone was inserted into the effluent tube. T h e glass collection tube was withdrawn on completion of the peak. The glass collection tube was rinsed with a few milliliters of acetone, the acetone concentrated to about 20 microliters and taken u p into a Hamilton microsyringe. The sample was then carefully added onto the prepared K B r in the disc holder according to the instructions of P a y n e and Cox (1966). T h e 1.5 mm. K B r micropellet was pressed and an infrared spect r u m obtained on a double beam Perkin Elmer infrared spectrophotometer (Model 457) equipped with 4x beam condenser, microsampling equipment, and die for 1.5 mm. and 0.5 mm. K B r pellets. RESULTS Verification of the Method. Reliability of the method for urinary estrogens in the female fowl was tested and the following results obtained. When known a m o u n t s of 3 H-labelled estrone, estradiol-17/3 and estriol were added to 24-hour aliquots of hen's urine before acid hydrolysis, the following means recoveries were obtained from three determinations prior to gas chromatography: E i = 61 + 2 % ; E 2 = 5 7 ± 5 % ; E 3 = 53 + 5 % . The precision of the technique was estimated in terms of the standard deviation, s, as derived from the expression of Snedecor (1956).
S = V £
d2/2N
where d is the difference between the results of each duplicate analysis and N is the n u m b e r of duplicate analyses. T h e observed value of s from six duplicate
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rated under nitrogen. T h e residue was taken up completely with several microliters of acetone in a Hamilton microsyringe for gas chromatography analysis. All analyses were conducted on F and M 810 analytical gas chromatograph equipped with a column oven with a programmed temperature control, flashheater, a flame ionization detector and electrometer. Column preparation were made according to Wotiz and Chattoraj (1964). Operating conditions were as follows: the column was set for programmed temperature operation 150-245°C, with a temperature increase of 20°C. per minute. (Though a programming rate of 6° per minute gave maximum peak heights for estrone acetate, 20° per minute was found suitable for rapid routine analysis). Detector and flash heater were set a t a temperature of 255°C. T h e carrier gas M2 and H2 were set at 40 ml. per minute to obtain maximum detector sensitivity. Under these conditions, retention times were E i A = 1 2 . 3 minutes, E 2 A = 1 4 . 8 minutes, E 3 A = 22.5 minutes, and E 4 A-24.4 minutes.
EDWARDS, JR.
69
TEMPERATURE EFFECTS ON ESTROGEN
analyses was 2.74 fig. of estrone per 24hour urine sample. This corresponded to a coefficient of variation of 8.3%. The lowest amount of estrogen detectable by flame ionization detector was 0.25 fig. estrone.
Several factors demonstrate the specificity of the method. The four urinary estrogen acetates, appearing as peaks on the gas chromatograph (Fig. 1), correspond exactly in retention times to those of reference estrogen acetates. These same Downloaded from http://ps.oxfordjournals.org/ at University of Victoria, McPherson Library Serials on April 26, 2015
4
8
12
16
20
24
28
MINUTES FIG. 1. Gas chromatographs of urinary estrogen acetates as compared with reference estrogen acetates.
70
F. Y. TANG, T. M. HUSTON AND H. M. EDWARDS, J R .
substances from the urine had twice previously been chromatographed on thin layer, once as the free estrogens and later as the acetate derivatives. These sub-
stances did not appear in blank determinations when distilled water was run through the entire procedure under identical conditions as the sample (Fig. 2).
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BLANK FRACTION 1
12
16
20
24
28
MINUTES FIG. 2. Gas chromatographs of a blank (distilled water carried through the entire analysis for urinary estrogen) as compared with reference estrogen acetates.
71
TEMPERATURE EFFECTS ON ESTROGEN
dOO
I 80 111
u z <
Estrone Acetate ..ws-V***
^U^w^w^'
Urine Fraction 1
3000
2500
2000 1800 1600 1400 1200 1000 800 FREQUENCY (CM" 1 )
600
400 250
4000 3500
3000
2500
2000 1800 1600 1400 1200 1000 800 600 400 250 FREQUENCY (CM' 1 )
FIG. 3. A comparison of the infrared spectra of estrone acetate and estriol triacetate against fractions one and three collected off the G.L.C.
Analyses of samples collected from the gas chromatograph showed that the labelled substances added to the urine to monitor recoveries were eluted at exactly the same retention times as reference estrogen acetates. Finally, infrared spectroscopy showed that the urinary estrone acetate collected from the gas chromatograph had an identical spectrum as that of reference estrone acetate (Fig. 3). I t can be seen from the various G.L.C. tracings that the TLC eluates are in general not pure; so that further separation of the estrogen under study is required. Gas chromatography under isothermal conditions was found to give inadequate separation of the estrogens from contaminant peaks. Use of temperature programming, however, allowed complete separation of individual estrogen peaks. Moreover, a flatter baseline was obtained
to facilitate more accurate quantitation. The only disadvantages encountered with temperature programmed gas chromatography were a slight overlapping of the estriol and 16-epiestriol peaks and somewhat more variable retention times, especially with the two estriols. Both difficulties were obviated by preliminary separation of the four estrogens prior to gas chromatography. The frequent presence of interfering materials in acid hydrolyzed urines appearing during gas chromatography necessitated the use of the second thin layer system (benzene-isopropyl ether 9:1) for the separation of the estrogen acetates. Rf values obtained by the T.L.C. system showed good reproductibility (EiA = .36 ±.05). From the results obtained in testing the method for accuracy, precision, sensitivity
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4000 3500
72
F. Y. TANG, T. M. HUSTON AND H. M. EDWARDS, JR.
TABLE 1 .—Twenty-four
Temperature
19°C.
Bird no.
1 2 3 4 5 6 7 8 9 10 11
Average Unoperated controls (av.) 30°C.
Average
hour urinary estrone excretion oj birds exposed to two different environmental temperatures Age in weeks 24
26
28
30
Age at first egg
g30.1* 23.7 25.3 36.5 76.2 6.5 24.5 27.6 20.2 30.5 16.0
g33.0 21.5 50.0 36.0 64.6 27.6 19.7 78.0 20.0 39.3 23.5
g65.0 20.0 27.7 38.2 23.5 5.0 11.5 35.7 20.0 15.1 32.5
g28.8 9.7 72.5 30.2 65.0 10.0 69.5 75.5 21.0 32.0 13.5
wks. 27 23 27 24 28 21 27 27 30 30 29
28.8
37.6
26.7
38.9
27
65.0 65.0 46.0 22.2 5.0 32.2 45.2 21.0 28.4 13.6 49.0
58.6 38.5 39.2 25.5 4.5 36.2 68.6 18.2 0.0 1.8 23.8
25.6 83.5 100.0 28.2 2.0 11.8 23.5 18.0 9.8 7.5 10.2
36.8 84.3 100.0 71.2 5.8 59.0 52.2 20.6 34.5 25.0 10.2
35.7
28.6
29.1
45.4
22 12 13 14 15 16 17 18 19 20 21 22
Unoperated controls (av.) Each urine sample represents a 24-hour collection period.
24 24 26 22 28 30
— 23 30 23 24
26 22
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24-hour value for urinary estrone either on or within a few days before the day of first lay. The mean biweekly excretion rate of urinary estrone (Table 3) was higher in the 30°C. environment at all ages except 26 weeks (Fig. 5). At this age, the higher mean value in the 19°C. environment was due to the high titers of urinary estrone from birds 3, 5 and 8, all of which were approaching sexual maturity, as well as the unusually low values of estrone of birds 20 and 21 (Table 1). Both values are considerably below the mean estrone excretion rate of both birds. These means were calculated from Table 1 to be 18.2
and specificity, it appears that thin layer and gas chromatography provide reliable chemical analysis for estrogens in fowl urine. Effects of Maturity and Environmental Temperature on Urinary Levels of Estrone and Estriol. As the birds approached sexual maturity, urinary titers of estrone and estriol increased (Fig. 4) in both environmental temperatures. There were generally sharp peaks of estrone and estriol around the day of first oviposition, though not always of maximum value for the individual bird (e.g. bird 100, 30°C.) The data in this respect agrees with Common et al. (1965) who reported a peak
73
T E M P E R A T U R E E F F E C T S ON ESTROGEN n~ Estrone | ' Estrlol
19°C
30°C 70
1
1
MM
III
1 1
II
to
to * IMS nit
1111
< • 'H
...* «
>0< 3
W
40 •
30 IO
10 0
n
ii
I
10 0
30
to
_.
3«
weeks #
1
1i o
a* is weeks
*
1*24
* 1S17 30
.
30
*
JC
i »
a *° S
30
w
10
*
»
5
a
1
0
s
1.•
it
# II
so
weeki
weeks
siaw
i
as
IHI
100
H'lHI H
eo so
so
TO
70
II
II
lltl l l l l l l l l l l l l I I I
III
16
30
40
so 40 30
ao 10
io M as weeks
so
0
weeks
FIG. 4. The relationship between urinary estrogen excretion and first egg production of birds at different ages in two different environmental temperatures. * Denote number of eggs beginning with first oviposition.
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-C 30
#88
so
It
74
c
F. Y. TANG, T. M. HUSTON AND H. M. EDWARDS, J R .
»
-1«°C
FIG. 5. Adjusted urinary estrone excretion at different ages in two environmental temperatures.
and 12.0 /*g./24 hours for birds 20 and 21, respectively. The volume of urine excreted per twenty-four hours does not appear to be of consequence, since both birds excreted greater than average 24-hour volumes of urine. Possible contributing factors for the extremely low values might be that bird 20 did not come into production until the end of the collection period (30 weeks), while bird 21, though laying nine eggs during the collection period, was not in production during the twenty-sixth week. Furthermore, the two birds gained only 56 and 38 grams, respectively, between the 24 and 26 weeks of age which is considerably less than the average weight gain of 123 grams for eleven birds at 30°C. for the same age period. The high values at 24 weeks of age (30°C.) and 26 weeks of age (19°C.) (Table 3) are due to the combined effect of birds approaching sexual maturity (birds 3, 5 and 8, 19°C, 26 weeks; birds 12, 13, and 22, 30°C, 24 weeks), (Table 1). There was no significant difference due to temperature, egg production, age of their interaction on either estrone or estriol excretion. There were highly signif-
TABLE 2.—Twenty-four hour urinary estriol excretion of birds exposed to two different environmental temperatures Age in weeks
Temperature
bird
19°C.
1 2 3 4 5 6 7 8 9 10 11
24
Average 30°C.
Average
12 13 14 15 16 17 18 19 20 21 22
26
28
30
10.0 6.0
0 0 5 5 5 8 5 3 2 10.1 5.6
g11.0 4.0 6.8 7.5 5.8 5.0 9.5 8.1 6.8 5.0 16.7
g0.0 4.0 8.3 5.0 0.0 6.2 10.0 8.5 6.8 7.2 5.0
7.0
8.4
7.8
5.5
16.5 10.0
6.8
7.0
10.5
12.5 10.5
7.1 6.7 7.2 5.2
g6.0* 5.5 5.2 15.8 15.0 0.0 6.7 7.2
9.0 5.0 8.5 7.5 5.7 7.5 0.0 7.2 6.8 7.6
8.8 4.5
4.5
10.2
17.2
7.5 8.7
5.7
11.9 10.7
6.5 0.0 5.0
0.0 6.8
9.0 9.2 —
11.2
10.8
6.0
6.0
7.2
8.8
11.9
10.7
15.4
* Each urine sample represents a 24-hour collection period.
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-30°C
icant (p<.01) differences observed between individual bird hormone levels. A correlation analysis run between the levels of estrone and estriol of birds in the two temperatures showed that at 19°C. there was a significant (p<.10 correlation between the two urinary estrogens, whereas at 30°C, the correlation coefficient (r) was not statistically significant. All birds held at 19°C. had a higher titer of estrone than estriol with a mean Ei:E3 ratio of 4.59 (Tables 1 and 2). The preponderance of estrone over the estriols agrees with most of the published reports (Mathur et al., 1966; Mulay et al., 1968). However, three birds held in the 30°C. environment deviated from this pattern and showed higher urinary estriol levels than estrone.
75
TEMPERATURE EFFECTS ON ESTROGEN
One bird (no. 16) in this environment showed higher urinary excretion of estriol than estrone throughout the period of study (Tables 1 and 2). Other birds (nos. 20 and 21) displayed higher estriol than estrone excretion at 26 and 28 weeks of age, respectively.
Urinary estrone and estriol were consistently measured quantitatively in 24 hour aliquots of maturing fowl urine. The levels of 17/3-estradiol and 16-epiestriol in such urine aliquots, however, were frequently below the sensitivity of the assay method. These do not agree with Mathur et al. (1966) who reported more 17j3-estradiol than estriol and more 16-espeistriol than estriol in laying hen's urine. Those differences may be due to differences in technique used. The average 24-hour urinary excretion rate of estrone and estriol was 33.0 and 7.2 fig. at 19°C. and 34.7 and 8.9 ng. at 30°C. (Table 3). These levels are considerably higher than those previously reported in the literature. The highest mean daily urinary estrone excretion value previously reported was 7.5 fig. three to five days before the resumption of lay. Mathur and Common (1967) estimated
TABLE 3.—Urinary estrogen excretion (g./24 hrs.) of birds at different ages in two different environmental temperatures
Treatment 19°C.
30°C.
1
No. of birds 11
11
Estrogen
Av.
S.E.
Estrone Estriol
28.8 7.7
Total
28 weeks
26 weeks
24 weeks 1
1
Av.
S.E.1
4.9 1.1
38.9 5.5
7.9 1.0
34.5
6.0
44.4
8.9
6.7 1.0
29.1 8.8
9.7 1.5
45.6 11.9
9.2 3.5
7.7
37.9
11.2
57.5
12.7
Av.
S.E.
5.3 1.5
37.6 8.4
5.8 1.2
26.7 7.8
36.5
6.8
46.0
7.0
Estrone Estriol
35.7 7.6
6.0 1.2
28.6 7.2
Total
43.3
7.2
35.8
Standard error of the mean (N = 11).
30 weeks
Av.
S.E.
1
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DISCUSSION
the estriol content of hen's urine as less than 1 ng. per day. Several factors may account for these differences. The combination of thinlayer and gas chromatography with its proven specificity by infrared analysis may be a more specific quantitative method than thin-layer and colorimetry. It is generally known that colorimetric techniques lack specificity and the presence of Kober inhibiting substances in acid hydrolyzed urine may lead to underestimated values. There is indirect evidence, from the levels of estrogen in fowl plasma, that the levels of urinary estrogen may be somewhat greater than that reported by Common et al. (1965). O'Grady (1968) has reported the plasma levels of estrone in the domestic fowl as of the order of 1.53.2 jug./lOO ml. This is within the range of 24-hour urinary estrone excretion as reported by Common et al. (1965). In human species, however, the urine concentration of estrone per 24-hours is of the order of 40-200 times the estrone concentration of 100 ml. of plasma. Assuming that the domestic fowl, likewise, concentrates estrogenic hormones in the urine, it seems appropriate to say that urinary levels of estrone should be several orders
76
F . Y. T A N G , T . M. H U S T O N AND H. M . E D W A R D S , J R .
Mulay, S., A. L. Carter and R. H. Common, 1968 Free and conjugated steroid estrogen fractions of hens' urine. Poultry Sci. 47: 67-76. O'Grady, J. E., 1968. The determination of oestraREFERENCES diol and oestrone in the plasma of the domestic Brown, J. B., 1955. A chemical method for the fowl by a method involving the use of labelled determination of oestriol, oestrone and oestradiol derivatives. Biochemistry J. 106: 77-86. in human urine. Biochemistry, 60: 185-193. Payne, W. R., Jr., and Wm. S. Cox, 1966. MicroCommon, R. H., L. Ainsworth, F. Hertelendy and infrared analysis of dieldrin, endrin, and other R. S. Mathur, 1965. The estrone content of hen's chlorinated pesticide residues in complex suburine. Canad. J. Biochemistry, 43: 539-547. strates. J. Assoc. Official Anal. Chem. 49: 989Lindner, H. R., 1959. Androgen in the bovine testis 996. and spermatic vein blood. Nature, 183: 1605- Snedecor, G. W., 1956. Statistical Methods. Iowa 1606. State College. Iowa Press, Ames, Iowa 5th ed. Lindner, H. R., and T. Mann, 1960. Relationship Subhas, T., and T. M. Huston, 1967. The influence between the content androgenic steroids in the of different environmental temperatures on the testes and secretory activity of the seminal vesplasma levels of FSH activity in female fowl. sicles in the bull. J. Endocrinology, 21: 341-372. Poultry Sci. 46: 1324. Mathur, R. S., P. A. Anastassiadis and R. H. ComVaron, H. H., H. A. Darnold, M. Murphy and J. mon, 1966. Urinary excretion of estrone and of 16Forsyth, 1967. Comparison of methods of detecepiestriol plus 17-epiestriol by the hen. Poultry tion for free estrogens and estrogen acetates on Sci. 45: 946-952. thin-layer chromatograms. Steroids, 9: 507-516. Wotiz, H. H., and S. C. Chattoraj, 1964. DeterMathur, R. S., and R. H. Common, 1967. Chromination of estrogens in low and high titer urine matographic identification of estriol and 16, 17using thin layer and gas liquid chromatography. epiestriol as constituents of the urine of the Anal. Chem. 36: 1466-1472. laying hen. Canad. J. Biochemistry, 45: 531-539. of m a g n i t u d e greater t h a n t h a t of fowl plasma.
L E O S. J E N S E N , R A L P H M A R T I N S O N AND G E O R G E SCHUMAIER
Department of A nimal Sciences, Washington State University, Pullman 99163 (Received for publication August 5. 1969)
D
ERMATITIS produced
in
turkey
b y feeding
poults
rations
cient in certain v i t a m i n s . A
is
defi-
dermatitis
this in poults fed diets sufficiently cient
to
reduce
growth
rate
defi-
(Heuser,
1935; a n d P a t r i c k et al., 1944). A defi-
characterized b y lesions on the corners of
ciency of biotin produced
the m o u t h and on t h e eyelids was ob-
which appeared
served in poults fed a diet deficient in
(Patrick et al., 1942). P a t r i c k et al. (1944)
p a n t o t h e n i c acid (Kratzer and Williams,
fed a purified diet deficient in either bio-
first
a
dermatitis
on the foot
pads
1948). A deficiency of riboflavin has also
tin or riboflavin
been reported to produce a dermatitis in
found t h a t dermatitis was prevented b y
turkey
Jukes,
biotin supplementation b u t not b y ribo-
1936), b u t others h a v e failed to observe
flavin. On t h e other h a n d , McGinnis a n d
1
poults
(Lepkovsky
and
Scientific Paper No. 3324, College of Agriculture, Washington State University, Pullman. Project No. 1941.
to turkey poults
and
Carver (1947) observed a dermatitis in poults fed a diet composed of practical ingredients which could be prevented by
Downloaded from http://ps.oxfordjournals.org/ at University of Victoria, McPherson Library Serials on April 26, 2015
A Foot Pad Dermatitis in Turkey Poults Associated with Soybean Meal 1
C. L. QUARLES, R. F. GENTRY AND G. 0 . BRESSLER
REFERENCES Andersen, A. A., 1958. New sampler for the collection sizing and conservation of viable airborne particles. J. Bact. 76: 471-484. Andersen, A. A., 1965. A sampler for respiratory health hazard assessment. Presented at the AIHA Conferences, May 6, 1965 at Houston, Texas. Andersen, A. A., and M. R. Andersen, 1961. A monitor for airborne bacteria. Applied Microbiol. 10: 181-184. Bressler, G. 0., 1961. Solar and windowless laying houses compared. Pennsylvania Agricultural Experiment Station, Science for the Farmer, Vol. IX, No. 2:3. Bressler, G. O., and A. J. G. Maw, 1966. High density and environmental control. Key to efficient egg production. Proceedings Thirteenth World's Poultry Congress, pages 468-473. Bressler, G. O., H. D. Bartlett and C. L. Quarles,
1967. Unpublished data at the Pennsylvania Experimental Station Farms. Chute, H. L., and M. Gershman, 1961. A new approach to hatchery sanitation. Poultry Sci. 40: 568-571. Chute, H. L., M. Gershman, R. M. Sherman and D. C. O'Meara, 1963. A program for sanitation for Maine chick hatcheries. Report Number 100, Maine Agricultural Experiment Station. Gentry, R. F., M. Mitrovic and G. R. Bubash, 1962. Application of Andersen sampler in hatchery sanitation. Poultry Sci. 41: 794-804. Gentry, R. F., 1968. Pennsylvania State University. Personal communications. Gentry, R. F., and C. L. Quarles, 1967. The measurement of bacterial contamination on egg shells. Poultry Sci. 46: 1263. Graves, R. C , and D. W. MacLaury, 1962. The effects of temperature, vapor pressure and absolute humidity on bacterial contamination of shell eggs. Poultry Sci. 41: 1219-1225. McNally, E. H., 1954. The relation of temperature and humidity to infection of shell eggs by bacteria. Section Papers of the 10th World Poultry Congress, page 107. Snedecor, G. W., 1956. Statistical Methods. The Iowa State University Press, Ames, Iowa. Williams, J. E., and A. D. Whittemore, 1967. A method for studying microbial penetration through the outer structures of the avian egg. Avian Diseases, 11: 467.
Effects of Different Environmental Temperatures on Urinary Estrogens of Maturing Fowl1 FRANK Y. TANG, TILL M. HUSTON AND HARDY M. EDWARDS, JR. Department of Poultry Science, University of Georgia, Athens, Georgia 30601 (Received for publication August 5, 1969)
T
HE principal estrogens of the domestic fowl are estrone, 17/3-estradiol estriol and 16-epiestriol (Mulay et al., 1968) Previous data (Mathur et al., 1966) has emphasized the significant differences in the ratios of urinary estrogens in the laying hen. The principal estrogens of the
1 University of Georgia, College of Agriculture, Experimental Stations Journal Series Paper 571, College Station, Athens, Georgia 30601.
domestic fowl, appearing in the urine in decreasing amounts, are estrone, 17^-estradiol, 16-espiestriol and estriol. It is not known, however, if there occurs any change in such ratios during the process of sexual maturation in the domestic fowl. In the prepubertal bull calf, for example, it has been shown (Linder, 1959; Linder and Mann, 1960) that androstenedion predominates while in adulthood, the re-
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5. Bacterial counts on the eggs produced in litter floor houses were 20 to 30 times greater than on eggs produced in wire floor houses. 6. The presence of coliform bacteria in embryos and chicks was related to their concentration in the air of the poultry house.
TEMPERATURE EFFECTS ON ESTROGEN
MATERIALS AND METHODS
Single Comb White Leghorn pullets (Kimber 137) raised in an uncontrolled ambient temperature were colostomized at 17 weeks of age. Subsequently, operated birds were separated at random into environmental temperature regimes of 19°C. and 30°C. with a diurnal cycle of 15-23°C. and 25-34°C. respectively. Four to five days following the operation, each colostomized bird was fitted with a self-retaining tygon cannulae inserted into the exteriorized rectum to prevent occlusion. Forty-eight hour urine collections were made from each modified bird at 24, 26, 28, and 30 weeks of age. To minimize bacterial action during the collection period, the collection apparatus was dusted with powdered penicillin G and dihydrostreptomycin sulfate. Urine aliquots were transferred every eight hours to individual quart-size containers, frozen immediately at — 15°C. pending chemical analysis. Estrogen Analysis. Analytical grade reagents were distilled in all-glass apparatus before use. To test for impurities, quantities of solvent corresponding in volume to the amount used during the procedure were evaporated to dryness and the residue injected with acetone into the gas chromatograph (Wotiz and Chattoraj, 1964). The same operation was performed
with petroleum ether (30-60°C. b.p.) after shaking with a 5:1 mixture of acetic anhydride :pyridine. In any perceptible peaks appeared corresponding in retention time to that of the estrogen acetates, the solvent was redistilled and the test procedure repeated. Estrogen analyses were made according to the procedure of Brown (1955) and Wotiz and Chattoraj (1964) with the following modifications. Twenty-four hour urine aliquots were filtered with suction through spun glass wool. Following acid hydrolysis, care was taken to wash the boiling chips thoroughly with diethyl ether in order to prevent sizeable losses due to absorption on the stones. The dried phenolic extract was subjected to thin layer chromatography along with reference standards in benzenejethyl acetate ( l : l v / v ) in a semi-saturated chamber-lining only the two side walls of the developing tank. After staining the reference standards with dichlorofluorescein and visualization under ultraviolet light, the corresponding areas of the urine sample were eluted individually with ethanol. Following acetylation, derivatives were chromatographed on thin layer with reference estrogen acetates, using benzene: isopropyl either (9:1) in an incompletely saturated chamber (Varon et al., 1967). Rf values for EiA=.36, E 2 A=.56, E3A = .30, and E
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verse case exists with testosterone predominating. Recently it has been shown by Subhas and Huston (1967) that environmental temperatures may alter the normal balance of gonadotrophic hormones in the maturing female fowl. The development of a sensitive and specific method of assay applicable to fowl urinary estrogens makes it possible to examine in greater detail the estrogen picture in the maturing domestic fowl and the influence of environmental temperature on estrogen.
67
68
F. Y. T A N G , T. M . H U S T O N AND H. M .
Recovery studies were made b y adding a measured quantity of tritium ( 3 H) labelled estrogen dissolved in absolute ethanol representing approximately 70,000 c.m.p. to a 24 hour sample or urine before acid hydrolysis. Aliquots were taken at various steps during the extraction procedure as a check on recoveries u p to gas chromatography. T h e aliquots were evaporated down to dryness in a scintillation vial. Then. 10 ml. scintillation fluid which contained 5 grams of 2,5-diphenyloxazole and l,4-bis-2(5-phenyloxazolyl)-benzene per liter of toluene was added, and the entire vial placed in a Packard Tricarb liquid scintillation spectrometer and counted for 5 minutes. T h e efficiency for tritium on this instrument was 29.0%. Corrections were made for background and quenching.
Collections of effluent from the gas chromatograph were accomplished b y means of a variable stream splitter beyond the column so t h a t a portion of t h e sample could be vented to the outside for collection. As the desired peak appeared, the capillary end of a glass collection tube containing a wad of glass wool soaked in acetone was inserted into the effluent tube. T h e glass collection tube was withdrawn on completion of the peak. The glass collection tube was rinsed with a few milliliters of acetone, the acetone concentrated to about 20 microliters and taken u p into a Hamilton microsyringe. The sample was then carefully added onto the prepared K B r in the disc holder according to the instructions of P a y n e and Cox (1966). T h e 1.5 mm. K B r micropellet was pressed and an infrared spect r u m obtained on a double beam Perkin Elmer infrared spectrophotometer (Model 457) equipped with 4x beam condenser, microsampling equipment, and die for 1.5 mm. and 0.5 mm. K B r pellets. RESULTS Verification of the Method. Reliability of the method for urinary estrogens in the female fowl was tested and the following results obtained. When known a m o u n t s of 3 H-labelled estrone, estradiol-17/3 and estriol were added to 24-hour aliquots of hen's urine before acid hydrolysis, the following means recoveries were obtained from three determinations prior to gas chromatography: E i = 61 + 2 % ; E 2 = 5 7 ± 5 % ; E 3 = 53 + 5 % . The precision of the technique was estimated in terms of the standard deviation, s, as derived from the expression of Snedecor (1956).
S = V £
d2/2N
where d is the difference between the results of each duplicate analysis and N is the n u m b e r of duplicate analyses. T h e observed value of s from six duplicate
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rated under nitrogen. T h e residue was taken up completely with several microliters of acetone in a Hamilton microsyringe for gas chromatography analysis. All analyses were conducted on F and M 810 analytical gas chromatograph equipped with a column oven with a programmed temperature control, flashheater, a flame ionization detector and electrometer. Column preparation were made according to Wotiz and Chattoraj (1964). Operating conditions were as follows: the column was set for programmed temperature operation 150-245°C, with a temperature increase of 20°C. per minute. (Though a programming rate of 6° per minute gave maximum peak heights for estrone acetate, 20° per minute was found suitable for rapid routine analysis). Detector and flash heater were set a t a temperature of 255°C. T h e carrier gas M2 and H2 were set at 40 ml. per minute to obtain maximum detector sensitivity. Under these conditions, retention times were E i A = 1 2 . 3 minutes, E 2 A = 1 4 . 8 minutes, E 3 A = 22.5 minutes, and E 4 A-24.4 minutes.
EDWARDS, JR.
69
TEMPERATURE EFFECTS ON ESTROGEN
analyses was 2.74 fig. of estrone per 24hour urine sample. This corresponded to a coefficient of variation of 8.3%. The lowest amount of estrogen detectable by flame ionization detector was 0.25 fig. estrone.
Several factors demonstrate the specificity of the method. The four urinary estrogen acetates, appearing as peaks on the gas chromatograph (Fig. 1), correspond exactly in retention times to those of reference estrogen acetates. These same Downloaded from http://ps.oxfordjournals.org/ at University of Victoria, McPherson Library Serials on April 26, 2015
4
8
12
16
20
24
28
MINUTES FIG. 1. Gas chromatographs of urinary estrogen acetates as compared with reference estrogen acetates.
70
F. Y. TANG, T. M. HUSTON AND H. M. EDWARDS, J R .
substances from the urine had twice previously been chromatographed on thin layer, once as the free estrogens and later as the acetate derivatives. These sub-
stances did not appear in blank determinations when distilled water was run through the entire procedure under identical conditions as the sample (Fig. 2).
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BLANK FRACTION 1
12
16
20
24
28
MINUTES FIG. 2. Gas chromatographs of a blank (distilled water carried through the entire analysis for urinary estrogen) as compared with reference estrogen acetates.
71
TEMPERATURE EFFECTS ON ESTROGEN
dOO
I 80 111
u z <
Estrone Acetate ..ws-V***
^U^w^w^'
Urine Fraction 1
3000
2500
2000 1800 1600 1400 1200 1000 800 FREQUENCY (CM" 1 )
600
400 250
4000 3500
3000
2500
2000 1800 1600 1400 1200 1000 800 600 400 250 FREQUENCY (CM' 1 )
FIG. 3. A comparison of the infrared spectra of estrone acetate and estriol triacetate against fractions one and three collected off the G.L.C.
Analyses of samples collected from the gas chromatograph showed that the labelled substances added to the urine to monitor recoveries were eluted at exactly the same retention times as reference estrogen acetates. Finally, infrared spectroscopy showed that the urinary estrone acetate collected from the gas chromatograph had an identical spectrum as that of reference estrone acetate (Fig. 3). I t can be seen from the various G.L.C. tracings that the TLC eluates are in general not pure; so that further separation of the estrogen under study is required. Gas chromatography under isothermal conditions was found to give inadequate separation of the estrogens from contaminant peaks. Use of temperature programming, however, allowed complete separation of individual estrogen peaks. Moreover, a flatter baseline was obtained
to facilitate more accurate quantitation. The only disadvantages encountered with temperature programmed gas chromatography were a slight overlapping of the estriol and 16-epiestriol peaks and somewhat more variable retention times, especially with the two estriols. Both difficulties were obviated by preliminary separation of the four estrogens prior to gas chromatography. The frequent presence of interfering materials in acid hydrolyzed urines appearing during gas chromatography necessitated the use of the second thin layer system (benzene-isopropyl ether 9:1) for the separation of the estrogen acetates. Rf values obtained by the T.L.C. system showed good reproductibility (EiA = .36 ±.05). From the results obtained in testing the method for accuracy, precision, sensitivity
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4000 3500
72
F. Y. TANG, T. M. HUSTON AND H. M. EDWARDS, JR.
TABLE 1 .—Twenty-four
Temperature
19°C.
Bird no.
1 2 3 4 5 6 7 8 9 10 11
Average Unoperated controls (av.) 30°C.
Average
hour urinary estrone excretion oj birds exposed to two different environmental temperatures Age in weeks 24
26
28
30
Age at first egg
g30.1* 23.7 25.3 36.5 76.2 6.5 24.5 27.6 20.2 30.5 16.0
g33.0 21.5 50.0 36.0 64.6 27.6 19.7 78.0 20.0 39.3 23.5
g65.0 20.0 27.7 38.2 23.5 5.0 11.5 35.7 20.0 15.1 32.5
g28.8 9.7 72.5 30.2 65.0 10.0 69.5 75.5 21.0 32.0 13.5
wks. 27 23 27 24 28 21 27 27 30 30 29
28.8
37.6
26.7
38.9
27
65.0 65.0 46.0 22.2 5.0 32.2 45.2 21.0 28.4 13.6 49.0
58.6 38.5 39.2 25.5 4.5 36.2 68.6 18.2 0.0 1.8 23.8
25.6 83.5 100.0 28.2 2.0 11.8 23.5 18.0 9.8 7.5 10.2
36.8 84.3 100.0 71.2 5.8 59.0 52.2 20.6 34.5 25.0 10.2
35.7
28.6
29.1
45.4
22 12 13 14 15 16 17 18 19 20 21 22
Unoperated controls (av.) Each urine sample represents a 24-hour collection period.
24 24 26 22 28 30
— 23 30 23 24
26 22
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24-hour value for urinary estrone either on or within a few days before the day of first lay. The mean biweekly excretion rate of urinary estrone (Table 3) was higher in the 30°C. environment at all ages except 26 weeks (Fig. 5). At this age, the higher mean value in the 19°C. environment was due to the high titers of urinary estrone from birds 3, 5 and 8, all of which were approaching sexual maturity, as well as the unusually low values of estrone of birds 20 and 21 (Table 1). Both values are considerably below the mean estrone excretion rate of both birds. These means were calculated from Table 1 to be 18.2
and specificity, it appears that thin layer and gas chromatography provide reliable chemical analysis for estrogens in fowl urine. Effects of Maturity and Environmental Temperature on Urinary Levels of Estrone and Estriol. As the birds approached sexual maturity, urinary titers of estrone and estriol increased (Fig. 4) in both environmental temperatures. There were generally sharp peaks of estrone and estriol around the day of first oviposition, though not always of maximum value for the individual bird (e.g. bird 100, 30°C.) The data in this respect agrees with Common et al. (1965) who reported a peak
73
T E M P E R A T U R E E F F E C T S ON ESTROGEN n~ Estrone | ' Estrlol
19°C
30°C 70
1
1
MM
III
1 1
II
to
to * IMS nit
1111
< • 'H
...* «
>0< 3
W
40 •
30 IO
10 0
n
ii
I
10 0
30
to
_.
3«
weeks #
1
1i o
a* is weeks
*
1*24
* 1S17 30
.
30
*
JC
i »
a *° S
30
w
10
*
»
5
a
1
0
s
1.•
it
# II
so
weeki
weeks
siaw
i
as
IHI
100
H'lHI H
eo so
so
TO
70
II
II
lltl l l l l l l l l l l l l I I I
III
16
30
40
so 40 30
ao 10
io M as weeks
so
0
weeks
FIG. 4. The relationship between urinary estrogen excretion and first egg production of birds at different ages in two different environmental temperatures. * Denote number of eggs beginning with first oviposition.
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-C 30
#88
so
It
74
c
F. Y. TANG, T. M. HUSTON AND H. M. EDWARDS, J R .
»
-1«°C
FIG. 5. Adjusted urinary estrone excretion at different ages in two environmental temperatures.
and 12.0 /*g./24 hours for birds 20 and 21, respectively. The volume of urine excreted per twenty-four hours does not appear to be of consequence, since both birds excreted greater than average 24-hour volumes of urine. Possible contributing factors for the extremely low values might be that bird 20 did not come into production until the end of the collection period (30 weeks), while bird 21, though laying nine eggs during the collection period, was not in production during the twenty-sixth week. Furthermore, the two birds gained only 56 and 38 grams, respectively, between the 24 and 26 weeks of age which is considerably less than the average weight gain of 123 grams for eleven birds at 30°C. for the same age period. The high values at 24 weeks of age (30°C.) and 26 weeks of age (19°C.) (Table 3) are due to the combined effect of birds approaching sexual maturity (birds 3, 5 and 8, 19°C, 26 weeks; birds 12, 13, and 22, 30°C, 24 weeks), (Table 1). There was no significant difference due to temperature, egg production, age of their interaction on either estrone or estriol excretion. There were highly signif-
TABLE 2.—Twenty-four hour urinary estriol excretion of birds exposed to two different environmental temperatures Age in weeks
Temperature
bird
19°C.
1 2 3 4 5 6 7 8 9 10 11
24
Average 30°C.
Average
12 13 14 15 16 17 18 19 20 21 22
26
28
30
10.0 6.0
0 0 5 5 5 8 5 3 2 10.1 5.6
g11.0 4.0 6.8 7.5 5.8 5.0 9.5 8.1 6.8 5.0 16.7
g0.0 4.0 8.3 5.0 0.0 6.2 10.0 8.5 6.8 7.2 5.0
7.0
8.4
7.8
5.5
16.5 10.0
6.8
7.0
10.5
12.5 10.5
7.1 6.7 7.2 5.2
g6.0* 5.5 5.2 15.8 15.0 0.0 6.7 7.2
9.0 5.0 8.5 7.5 5.7 7.5 0.0 7.2 6.8 7.6
8.8 4.5
4.5
10.2
17.2
7.5 8.7
5.7
11.9 10.7
6.5 0.0 5.0
0.0 6.8
9.0 9.2 —
11.2
10.8
6.0
6.0
7.2
8.8
11.9
10.7
15.4
* Each urine sample represents a 24-hour collection period.
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-30°C
icant (p<.01) differences observed between individual bird hormone levels. A correlation analysis run between the levels of estrone and estriol of birds in the two temperatures showed that at 19°C. there was a significant (p<.10 correlation between the two urinary estrogens, whereas at 30°C, the correlation coefficient (r) was not statistically significant. All birds held at 19°C. had a higher titer of estrone than estriol with a mean Ei:E3 ratio of 4.59 (Tables 1 and 2). The preponderance of estrone over the estriols agrees with most of the published reports (Mathur et al., 1966; Mulay et al., 1968). However, three birds held in the 30°C. environment deviated from this pattern and showed higher urinary estriol levels than estrone.
75
TEMPERATURE EFFECTS ON ESTROGEN
One bird (no. 16) in this environment showed higher urinary excretion of estriol than estrone throughout the period of study (Tables 1 and 2). Other birds (nos. 20 and 21) displayed higher estriol than estrone excretion at 26 and 28 weeks of age, respectively.
Urinary estrone and estriol were consistently measured quantitatively in 24 hour aliquots of maturing fowl urine. The levels of 17/3-estradiol and 16-epiestriol in such urine aliquots, however, were frequently below the sensitivity of the assay method. These do not agree with Mathur et al. (1966) who reported more 17j3-estradiol than estriol and more 16-espeistriol than estriol in laying hen's urine. Those differences may be due to differences in technique used. The average 24-hour urinary excretion rate of estrone and estriol was 33.0 and 7.2 fig. at 19°C. and 34.7 and 8.9 ng. at 30°C. (Table 3). These levels are considerably higher than those previously reported in the literature. The highest mean daily urinary estrone excretion value previously reported was 7.5 fig. three to five days before the resumption of lay. Mathur and Common (1967) estimated
TABLE 3.—Urinary estrogen excretion (g./24 hrs.) of birds at different ages in two different environmental temperatures
Treatment 19°C.
30°C.
1
No. of birds 11
11
Estrogen
Av.
S.E.
Estrone Estriol
28.8 7.7
Total
28 weeks
26 weeks
24 weeks 1
1
Av.
S.E.1
4.9 1.1
38.9 5.5
7.9 1.0
34.5
6.0
44.4
8.9
6.7 1.0
29.1 8.8
9.7 1.5
45.6 11.9
9.2 3.5
7.7
37.9
11.2
57.5
12.7
Av.
S.E.
5.3 1.5
37.6 8.4
5.8 1.2
26.7 7.8
36.5
6.8
46.0
7.0
Estrone Estriol
35.7 7.6
6.0 1.2
28.6 7.2
Total
43.3
7.2
35.8
Standard error of the mean (N = 11).
30 weeks
Av.
S.E.
1
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DISCUSSION
the estriol content of hen's urine as less than 1 ng. per day. Several factors may account for these differences. The combination of thinlayer and gas chromatography with its proven specificity by infrared analysis may be a more specific quantitative method than thin-layer and colorimetry. It is generally known that colorimetric techniques lack specificity and the presence of Kober inhibiting substances in acid hydrolyzed urine may lead to underestimated values. There is indirect evidence, from the levels of estrogen in fowl plasma, that the levels of urinary estrogen may be somewhat greater than that reported by Common et al. (1965). O'Grady (1968) has reported the plasma levels of estrone in the domestic fowl as of the order of 1.53.2 jug./lOO ml. This is within the range of 24-hour urinary estrone excretion as reported by Common et al. (1965). In human species, however, the urine concentration of estrone per 24-hours is of the order of 40-200 times the estrone concentration of 100 ml. of plasma. Assuming that the domestic fowl, likewise, concentrates estrogenic hormones in the urine, it seems appropriate to say that urinary levels of estrone should be several orders
76
F . Y. T A N G , T . M. H U S T O N AND H. M . E D W A R D S , J R .
Mulay, S., A. L. Carter and R. H. Common, 1968 Free and conjugated steroid estrogen fractions of hens' urine. Poultry Sci. 47: 67-76. O'Grady, J. E., 1968. The determination of oestraREFERENCES diol and oestrone in the plasma of the domestic Brown, J. B., 1955. A chemical method for the fowl by a method involving the use of labelled determination of oestriol, oestrone and oestradiol derivatives. Biochemistry J. 106: 77-86. in human urine. Biochemistry, 60: 185-193. Payne, W. R., Jr., and Wm. S. Cox, 1966. MicroCommon, R. H., L. Ainsworth, F. Hertelendy and infrared analysis of dieldrin, endrin, and other R. S. Mathur, 1965. The estrone content of hen's chlorinated pesticide residues in complex suburine. Canad. J. Biochemistry, 43: 539-547. strates. J. Assoc. Official Anal. Chem. 49: 989Lindner, H. R., 1959. Androgen in the bovine testis 996. and spermatic vein blood. Nature, 183: 1605- Snedecor, G. W., 1956. Statistical Methods. Iowa 1606. State College. Iowa Press, Ames, Iowa 5th ed. Lindner, H. R., and T. Mann, 1960. Relationship Subhas, T., and T. M. Huston, 1967. The influence between the content androgenic steroids in the of different environmental temperatures on the testes and secretory activity of the seminal vesplasma levels of FSH activity in female fowl. sicles in the bull. J. Endocrinology, 21: 341-372. Poultry Sci. 46: 1324. Mathur, R. S., P. A. Anastassiadis and R. H. ComVaron, H. H., H. A. Darnold, M. Murphy and J. mon, 1966. Urinary excretion of estrone and of 16Forsyth, 1967. Comparison of methods of detecepiestriol plus 17-epiestriol by the hen. Poultry tion for free estrogens and estrogen acetates on Sci. 45: 946-952. thin-layer chromatograms. Steroids, 9: 507-516. Wotiz, H. H., and S. C. Chattoraj, 1964. DeterMathur, R. S., and R. H. Common, 1967. Chromination of estrogens in low and high titer urine matographic identification of estriol and 16, 17using thin layer and gas liquid chromatography. epiestriol as constituents of the urine of the Anal. Chem. 36: 1466-1472. laying hen. Canad. J. Biochemistry, 45: 531-539. of m a g n i t u d e greater t h a n t h a t of fowl plasma.
L E O S. J E N S E N , R A L P H M A R T I N S O N AND G E O R G E SCHUMAIER
Department of A nimal Sciences, Washington State University, Pullman 99163 (Received for publication August 5. 1969)
D
ERMATITIS produced
in
turkey
b y feeding
poults
rations
cient in certain v i t a m i n s . A
is
defi-
dermatitis
this in poults fed diets sufficiently cient
to
reduce
growth
rate
defi-
(Heuser,
1935; a n d P a t r i c k et al., 1944). A defi-
characterized b y lesions on the corners of
ciency of biotin produced
the m o u t h and on t h e eyelids was ob-
which appeared
served in poults fed a diet deficient in
(Patrick et al., 1942). P a t r i c k et al. (1944)
p a n t o t h e n i c acid (Kratzer and Williams,
fed a purified diet deficient in either bio-
first
a
dermatitis
on the foot
pads
1948). A deficiency of riboflavin has also
tin or riboflavin
been reported to produce a dermatitis in
found t h a t dermatitis was prevented b y
turkey
Jukes,
biotin supplementation b u t not b y ribo-
1936), b u t others h a v e failed to observe
flavin. On t h e other h a n d , McGinnis a n d
1
poults
(Lepkovsky
and
Scientific Paper No. 3324, College of Agriculture, Washington State University, Pullman. Project No. 1941.
to turkey poults
and
Carver (1947) observed a dermatitis in poults fed a diet composed of practical ingredients which could be prevented by
Downloaded from http://ps.oxfordjournals.org/ at University of Victoria, McPherson Library Serials on April 26, 2015
A Foot Pad Dermatitis in Turkey Poults Associated with Soybean Meal 1