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Photoperiods and age as factors modifying the effects of Kepone in Japanese quail
TOXICOLOGY
AND
APPLIED
Photoperiods
PHARMACOLOGY
29,329-339
(1974)
and Age as Factors Modifying Kepone in Japanese Quail VICTORP. EROSCHENKO' AND
the Effects
of
WILBOR0. WILSON
Department of Anatomy, School of Veterinary Medicine, and Department of Avian Sciences, University of California, Davis, California 95616 Received August 14,1973; accepted March 21,1974
Photoperiods and Age as Factors Modifying the Effects of Kepone in JapaneseQuail. EROSCHENKO, V. P. AND WILSON, W. 0. (1974). Toxicol. Appl. Pharmacol. 29, 329-339.Immature Japanese quail (Coturnix coturnix japonica) of both sexesfed 200 ppm of the insecticideKepone, whenexposedto 16hr of light per day, hadsignificantly enlargedreproductive organs,livers and adrenalglands.After a reduction in the photoperiod high mortality occurred in only the birds previously fed Kepone. Administration of Keponeto adult femaleswhile egg-layingdid not affect the weight of reproductive organsor livers. In adult malesof the sameagetestesand livers weregreatly enlarged.In both sexesof adult birds, the adrenalswere enlarged.Subsequentexposureto reducedphotoperiod of the Kepone-fed adult quails of both sexesproduced no mortality. In malesfed Kepone, testicular regressionwasrecordedmuch earlier than in controls. Previous ingestionof Kepone affected neither the cessationof egg-layingnor the regressionof the ovary. Keponeexertedits effectsprimarily on the immature quail of both sexesand to a lesserdegreeon adults. An estrogenicaction of Kepone wasindicated by increasedoviduct weight of young femaleson a photoperiod of 6 hr light and 18hr dark per day. In adult malesthe major estrogeniceffect wasincreasedliver weight. Kepone’ (decachlorotetracyclodecanone) is a complex polycyclic chlorinated insecticide of high stability that has been found useful in controlling many speciesof insects,including roaches, ants, wireworm and leaf-eaters, aswell asfly larvae (Epstein and Legator, 1971; Brown, 1966). However, Kepone has been reported to interfere with the reproduction of mammals and birds and to affect various organs of the body. Various concentrations of Kepone in the diet reduced reproduction in mice (Huber, 1965), bobwhite quail, and pheasants (Dewitt et al., 1962, 1963) as well as decreased egg production in laying chickens (Naber and Ware, 1965).
Furthermore, Kepone has been shown to exert or promote “estrogenic” activity in the Japanese quail or common coturnix. Administration of Kepone to immature, hypophysectomized and ovariectomized quail resulted in an increase of the oviduct weight. Also, intact Kepone-fed quail exhibited enlarged ovaries characterized by a greater number of developing follicles, whereas Kepone-treated males had either
atrophic or greatly enlarged testes. An effect of Kepone that was common to both sexes of quail was an enlargement of the liver (McFarland and Lacy, 1969). 1Presentaddress:Departmentof BiologicalSciences, Universityof Idaho, Moscow,Idaho 83843. z Suppliedby Agricultural Divisionof Allied Chemicals, NewYork. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
329
330
EROSCHENKO
AND
WILSON
The present study was undertaken to compare the weekly responses of various organs of the Japanese quail (Coturnix coturnix japonica) due to gonadotropins produced by photostimulation with the estrogenlike effects produced by feeding the insecticide Kepone. METHODS Immature and mature individuals of each sex from the University of California, Davis coturnix Random Line 908 were used in the experiments. The eggs were hatched in a forced-draft, single-stage incubator, and the chicks were brooded in commercial batteries with temperature and humidity regulated as described by Woodard et al. (1965) and Wilson et al. (1962). At 3 weeks of age the birds were separated according to sex based on plumage dimorphism, placed in individual wire-floor cages and exposed to 16 hr of light and 8 hr of darkness (16L : 8D) ; previously they were given continuous light (24L). The intensity of light was 30 foot-candles and was from an incandescent source. The ambient temperature of the room was kept at 22” rt 1“C. Birds of each sex were separated into control and experimental groups. The control group of birds was given turkey starter ration and water ad libitum. The experimental group was given the same diet except that 200 ppm of Kepone had been added. To ensure proper distribution, Kepone was first dissolved in acetone, thoroughly mixed into the feed, and then administered to the various experimental groups at the designated time. In experiments 1 and 3 the reduction of photoperiod occurred after Kepone administration. In experiment 2, the birds were allowed to mature under the reduced photoperiod prior to being fed Kepone (Fig. 1). After the reduction in the daily photoperiod the egg-laying performance of females from both the experimental and control groups of experiment 3 was recorded daily in order to establish the pattern of the cessation of egg laying. After egg laying had ceased groups of varying numbers of birds were sacrificed as scheduled. At the time of sacrifice in all the experiments, organs from both sexes of birds during both the stimulatory and inhibitory light regimens were excised and the weights of the ovary, oviduct, testes, liver, adrenal glands and carcass were taken on a Mettler balance and recorded. The oviduct was dissected, uncoiled and its length was measured to the nearest centimeter. All organs were fixed in formalin solution and prepared for subsequent histologic examination. The details of the designs for all 3 experiments are illustrated in Fig. 1. For statistical treatment of the data the Student t test was used to evaluate differences between means of the experimental and control group of the same age. A p value greater than 0.05 was not considered significant; however, when the probability for a given comparison was less than 0.05, these probabilities were listed. RESULTS Experiment
1
By the end of the first week of Kepone feeding a significant weight increase was recorded in the oviducts, adrenals and livers, whereas an increase in the weight of testes and ovaries was not significant until after the second and third week of Kepone feeding (Table 1). The reduction in light at 6 weeks of age from 16L : 8D to the 6L : 18D regimen had a severe effect on the survival rate of both sexes only when fed Kepone.
KEPONE EFFECTS IN QUAIL EXP.
331
I X
4
6 AGE
EXP.
10
8
(weeks)
2
AGEtweeks)
EXP.
3
x
0 E t E
--
x
x
x
X
t
6
2
II
24 AGE
26
2a
30
(weeks)
FIG. 1. Experimental design of three experiments with different photoperiodic treatment. The ages are on the abscissa: Key: X = Termination schedule of the quail during the experiments. K = Duration of feeding Kepone (200 ppm) to experimental group (both sexes) is shown within the arrows.
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EROSCHENKO
AND
WILSON
The immature males were most severely affected as 15 out of 21 died within the average time of 4.3 days, with a range from 1 to 9 days. Females were less severely affected; 10 of 17 died. The average time of death and range for the immature females was almost the same as for the males. The common characteristic in the dead birds of both sexes was a severe loss of total body weight. All experimental birds that were dying exhibited severe body tremors and ataxia. At the onset of body tremors there was no indication that birds consumed food. The result of postmortem examination indicated the birds that died earlier had heavier body weights. Birds that died at a later date were without visible body fat and had severely emaciated pectoral musculature. Kepone-fed birds of both sexes lost about 40-50 g of their total body weight within the mean time of 4.3 days prior to death. Postmortem examination of various organs indicated that the mean liver, testes, ovarian and oviducal weight of the dead birds was significantly less than the corresponding mean organ weight from the birds terminated just prior to light reduction. TABLE 1 EFFECT LENGTH
OF INSECTICIDE KEPONE OR WEIGHT OF ORGANS
Parameters measured Kepone treatment duration (weeks) Oviduct length (cm) Control Kepone fed Oviduct wt (g) Control Kepone fed Ovarian wt (g) Control Kepone fed Testes wt (g) Control Kepone fed Adrenal wt (mg) Control Kepone fed Control Kepone fed Liver wt (g) Control Kepone fed Control Kepone fed a (0) = Number b p < 0.05. cp < 0.01.
dp < 0.001.
of birds.
OF 16L: SD ON THE MEAN OF BOTH SEXES IN EXPERIMENT 1
(200 PPM) AND A PHOTOPERIOD IN YOUNG
Sex
COTURNIX
3 0
Age of birds (weeks) --____-~~~~ 5 -
1
6
__~---
2
3 13.3 (10) 24.2 (7)
F F
3.6 (7) -
3.7 (7) 8.4 (7)d
6.5 (8) 14.8 (7)d
F F
0.02 (7) -
0.03 (7) 0.68 (7)d
0.45(8) 2.02 (7)d
2.57 (10) 4.96(7)b
F F
0.04(7) -
0.05(7) 0.08 (7)
0.09(8) 0.17(7)
6.82 (7)d
M M
0.10 (7) -
0.21 (7) 0.34(7)
0.49 (7) 1.57 (7)
1.59(7) 2.72 (8)"
F F M
8.60(7)
9.24(7) 10.71 (7)b 9.66(7) 12.56(7)b
10.39(8) 14.69(7) 12.12 (7) 15.29(7)
12.81 (10)
M
7.2;(7) -
F F M M
2.72(7) 2.37 (7) -
3.60(7) 4.93 (7) 3.02 (7) 4.33 (7)d
3.19(8) 6.63 (7)d 3.19 (7) 4.51(7)
1.44 (10)
18.36 (7) 11.07 (7) 17.36 ( 8)d
3.90 6.74 2.63 7.75
(10)
(7)d (7) (8)d
KEPONE
EFFECTS
333
IN QUAIL
On the other hand, the mean weight of the adrenal glands from both sexes of birds that died remained unchanged. The birds of both sexes that survived the initial 7 to 9 days of light reduction did not show body tremors, continued to eat food and appeared normal. The surviving birds were observed daily for an additional 4 weeks after the light change and were terminated when 10 weeks of age. None of the control birds died after the light was reduced. Experiment 2
After 1 week of the Kepone-containing diet significant weight changes were observed in the oviducts and livers, whereas the weight of adrenal glands did not change significantly until after the second and third week of Kepone feeding (Table 2). After 3 weeks of treatment with Kepone, the ovarian weights were 37 and 30 mg and testicular weights were 16 and 13 mg for the experimental and control groups, respectively. Thus under reduced photoperiods the gonads were not significantly altered during the entire 3 weeks of Kepone treatment. No observable body tremors, ataxia or death were recorded in any Kepone-treated or control birds of either sex. TABLE 2 EFFECT
OF INSECTICIDE KEPONE (200 ppm) AND A PHOTOPERIOD LENGTH AND WEIGHT OF ORGANS IN COTURNIX OF BETH
OF 6L: 18D ON THE MEAN SEXES IN EXPERIMENT 2
Age of birds (weeks) Parameters measured Kepone treatment duration (weeks) Oviduct length (cm) Control Kepone fed Oviduct wt (g) Control Kepone fed Liver wt (g) Control Kepone fed Control Kepone fed Adrenal wt (mg) Control Kepone fed Control Kepone fed
Sex
6
7
8
9
0
1
2
3
F F
3.4 (7) -
3.6 (7) 9.2 (7)
3.6 (7) 12.7 (7)
3.9 (7) 15.8 (7)
F F
0.02 (7) -
0.02 (6) 0.72 (7)
0.02 (7) 1.19 (7)
0.03 (7) 1.60 (7)
F F M M
2.72 (7)
2.94 (7) 4.69 (7)’ 2.65 (8) 3.67 (7)*
3.01 (7) 6.25 (7) 2.62 (7) 4.44 (7)”
2.84 (7) 6.41 (7) 2.27 (7) 5.95 (7)
F F M M
10.60 (7)
9.70 (7) 10.80 (7) 9.91 (7) 12.60 (7)b
9.30 (7) 14.50 (7) 10.40 (7) 12.40 (7)b
3.10 (8) -
l&8) -
11.20 (7) 12.20 (7) 8.50 (8) 9.62 (7)
a (0) = Number of birds. *p < 0.01. ep < 0.001.
Experiment 3
Neither 3 weeks of Kepone administration to egg-laying quail nor 2 weeks of reduced photoperiod (6L: 1SD) produced any significant changes in the mean oviduct length,
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EROSCHENKO
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nor in the oviduct, ovarian, and liver weight. But, after 3 weeks of reduced light significant changes were recorded in the mean oviduct length, oviduct and liver weight. The ovaries were completely unaffected during the Kepone feeding or after the reduction of photoperiod (Table 3), whereas the adrenal glands were significantly heavier only at the end of 3 weeks of Kepone feeding. Daily observation, after reduction of light, demonstrated that the average time required for cessation of egg-laying in both the control and experimental birds was 8.0 and 8.3 days, respectively, and was consequently statistically insignificant. Three weeks of Kepone feeding to adult males resulted in significant weight increases in the adrenal gland, liver and testes. Reduction of the photoperiod for 1 week did not TABLE EFFECT
OF INSECTICIDE MEAN WEIGHT
3
KEPONE (200 ppm) DURING REDUCED PHOTOPERIODS ON LENGTH OF ORGANS IN SEXUALLY MATURE COTURNIX IN EXPERIMENT 3
AND
Age of birds (weeks) Parametersmeasured Sex
27
28
29
30
31
Length without Kepone (weeks) after reduction of light to 6L: 18D:
0
1
2
3
4
F
25.6 (7) 26.5 (7)
Laying Laying
10.5 (11) 10.5 (11)
8.3 (12) 9.7 (1l)b
7.3 (9) 8.3 (9)
F F
6.37 (7) 6.29 (7)
Laying Laying
0.74 (11) 0.82 (11)
0.37 (12) 0.51 (1l)b
0.27 (9) 0.34 (9)
F F
6.21, (7) 5.45 (7)
Laying Laying
0.12 (11) 0.12 (11)
0.10 (12) 0.11 (11)
0.07 (9) 0.09 (10)
M M
2.73 (7) 3.86 (7)
2.28 (12) 1.06 (12)”
0.39 (12) 0.31 (9)
0.09 (10) 0.02 (12)b
0.05 (7) 0.03 (13)
ll.oo(ll) 12.20(11) 10.61(12) 13.31(1l)b
Oviduct length (cm) Control Kepone fed Oviduct wt (g) Control Kepone fed Ovarian wt (g) Control ‘Keponefed Testeswt (g) Control Kepone fed Adrenal wt (mg) Control Keponefed Control Kepone fed Liver wt (g) Control Kepone fed Control Kepone fed
F
F F M M
12.91 16.50 11.52 15.30
(7) (7)b (7) (7)b
Laying Laying 11.30(12) 12.90(12)
F F M M
6.34 6.88 2.59 5.35
(7) (7) (7) (7)d
Laying Laying 2.84 (12) 6.24 (12)d
1 Ly)O=OJumber of b’lr d s’, 1aying females were not sampled. =p < 0:01: dp < 0.01.
3.71 3.67 3.18 5.72
(11) (11) (12) (9)d
12.00 13.31 11.42 13.91
(12) (11) (10) (12)b
11.40 11.90 12.01 12.13
(9) (9) (7) (13)
3.42 4.24 3.29 6.28
(12) (1 l)b (10) (12)b
3.42 4.40 2.62 4.29
(9) (10)b (7) (1 3)d
KEPONE
EFFECTS
IN QUAIL
335
affectthemeantesticularweightin thecontrolbirds.However,thetestesfromKeponefed birds were significantly smaller than those from the control birds after 1 and 3 weeksof reducedphotoperiod. The meanliver weight from experimental birds remained significantly heavier during the entire 4 weeks of reduced light, whereas the adrenal glands were significantly heavier during various intervals after the light reduction (Table 3). The reduction of light at 27 weeks of age did not produce any body tremors, ataxia, visible ill-effects or mortality in either the Kepone-fed or control group birds of either sex. DISCUSSION Increases in both weight and length of the oviduct in the immature control birds under stimulatory light was initially recorded after the 4th week of age. However, the oviducts in the immature quail subjected to a reduced light cycle of 6L : 18D remained undeveloped, A light regimen of 16L:8D was shown to be stimulatory to gonadal development of young chicks of either sex. Under these light conditions females produced eggs at 7 to 8 weeks of age and spermatogenic activity was see%in males at about 7 weeks of age. Decrease in daylight from 18 to 6 hr delayed or inhibited sexual maturation and was nonstimulatory to the gonads (Wilson et al., 1962; Mather and Wilson, 1964; Follett and Farner, 1966; Tanaka et al., 1965). Reports indicate that the oviduct of immature avian species differentiates rapidly when subjected to exogenous hormones. Exogenous estrogenic compounds administered to immature chicks produced an increase in the oviduct weight and development of tubular glands (Oka and Schimke, 1969a, b; Palmiter and Wrenn, 1971; Jackson et al., 1971). Certain insecticides exert estrogenic effect on the reproductive organs of birds. Treatment of immature chicks and Japanese quail with o,p’-DDT produced enlarged oviducts and increased glycogen content of the oviducts (Bitman et al., 1968; Cooke, 1970). Administration of Kepone to sexually immature chickens likewise produced enlarged oviducts (Sherman and Ross, 1961). The insecticide Kepone exerted a highly stimulatory effect on the oviducts of sexually immature quail. Accelerated growth in length and weight of the oviduct was recorded within the first 2 weeks of Kepone administration. The most pronounced effect was noted in the oviducts of immature quail subjected to reduced (6L : 18D) light regimen. The oviducts were enlarged more than 60 times over the controls. Histology of the oviducts in the regions of the magnum, isthmus and uterus showed cellular hypertrophy, hyperplasia, ciliated surface epithelium, formation of tubular glands and accumulation of secretory material in these cells (submitted for publication). In laying quail, on the other hand, Kepone ration did not alter the weight or the length of the oviduct. Histologic examination of these oviducts did not indicate any detectable cellular alterations. Similar results were obtained when Kepone ration was given to female quail just beginning to lay eggs. Thus it appears that Kepone exerted an “estrogenlike” effect on the undeveloped oviducts of only maturing quail, whereas no additional effect was seen in the fully functional oviduct of the laying quail. In experiments with ovariectomized and hypophysectomized quail, McFarland and
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EROSCHENKOAND WILSON
Lacy (1969) postulated that Kepone could stimulate the hypothalamic-pituitary axis to release: FSH and thereby stimulate ovarian estrogen release as well as direct stimulation of the immature oviduct in the absence of the pituitary. The greatest estrogenlike effect of Kepone occurred in quail with the intact pituitary. Kepone produced changes neither in the ovarian weight nor in egg production in the laying quail. Furthermore, Kepone did not affect the developing ovary in the maturing quail exposed to stimulatory light until after the fifth week of age. The ovaries were characterized by 3 to 4 larger follicles and a greater number of smaller developing follicles. McFarland and Lacy (1969) reported a reduced number of eggs in the uterus of Kepone-fed quail, thus implying a reduced egg production. In Kepone-treated adult mice, normal follicular development with few corpora lutea was recorded, Hormone bioassay showed that the mice were under prolonged stimulation of FSH and estrogen but under insufficient LH stimulation. This blocking of rhythmic release of LH by Kepone could prevent ovulation and corpora lutea formation in mice (Huber, 1965). The effect of Kepone on the ovaries of maturing quail exposed to stimulatory light could be similar to that reported in mice by Huber (1965), whereas its effect on the quail oviduct could have a dual action as postulated by McFarland and Lacy (1969). The increase inpviduct size, weight and cellular differentiation that we found during the reduced light (6L: 18D) and the absence of increased ovarian weight strongly suggests a direct estrogenic action of Kepone on the oviduct. Quail of both sexes fed Kepone had enlarged adrenals. The heaviest adrenals were recorded in the birds that were undergoing sexual maturation under stimulatory light. Due to such increases in adrenal gland size in all Kepone-fed birds, it was highly probable that the action of Kepone was similar to that of an internal stressor and the response of the adrenals followed the classical concept of changes associated with stress (Selye, 1950). Kepone administration to maturing and adult males under a 16L:8D light cycle produced heavier testes. The testes appeared edematous and histologic examination revealed a great distention of seminiferous tubules, apparently containing fluid (submitted for publication). Also Kepone appeared to exert its estrogenlike action on the testes by reducing their weight below that of controls after the first week of light reduction. It has been reported that physiologic doses of estrogen or prolactin reduces weight of enlarged testes, depresses spermatogenesis and inhibits gonadal growth (Van Tienhoven, 1961; Meier et al., 197 1). It has been also shown that testes enlargement occurred in unstressed chicks after deoxycorticosterone acetate (DCA), cortisone and ACTH administration (Conner, 1959). Histologic appearance of testes from DCAtreated chicks showed a dilated seminiferous tubule system, and a stretched and flattened germinal epithelium (Boas, 1958). Furthermore, high salt diet given to immature chicks produced hypertrophy of the seminiferous tubules and later transformed them into thin-walled cysts containing water-clear fluid (Siller et al., 1972). The insecticide Kepone likewise produced increased testicular weight and an associated distention of seminiferous tubules by accumulation of waterlike fluid. An important observation in this study was the fact that a reduction of light to 6 hr per 24 hr at 6 weeks of age produced a very high mortality rate in the Kepone-fed quail of both sexes. Young males were affected more than the females although the mean time of death and the signs of intoxication were similar to those reported in mice
KEPONE EFFECTSIN QUAIL
331
(Huber, 1965), in chickens (Sherman and Ross, 1961), in pheasants (Azavedo et al., 1965) and in cowbirds (Stickel etal., 1970). Decreased weights of body carcass, reproductive organs, absence of visible fat stores and emaciation were the striking features observed in the birds that died. Similar light reduction failed to produce either the signs of intoxication or mortality in Kepone-fed adult quail of either sex. Also, feeding Kepone during the reduced light to 6-week-old birds likewise did not cause mortality. Since death in the younger birds of both sexes occurred after prolonged tremors and since there was a high weight loss, it was possible that available fat stores were mobilized at this time. Chlorinated hydrocarbon pesticides are fat soluble and have been shown to accumulate in fatty tissue of hens and in the egg yolk (Smith et al., 1970). In laying hens fed different concentrations of Kepone, egg yolk residues of Kepone reached a maximum by the fifth week (Naber and Ware, 1964). Other chlorinated pesticides such as DDT were shown to accumulate in egg yolk of chickens and Japanese quail in proportion to dietary levels of DDT (Smith et al., 1969, 1970). Furthermore, 10% or more of the daily intake of DDT in pheasants can be excreted by laying females in their eggs, thus reducing the quantity of ingested DDT placed into other fat deposits (Azavedo et al., 1965). Since the chlorinated insecticides are stored in the body fat, the mobilization of this stored insecticide would have an effect on the well being of the bird. Mortality is most likely to occur at times of seasonal or emergency weight loss and during other stresses, for then the stored toxicant is released into blood and other tissues causing toxicity (Ecobichon and Saschenbrecker, 1968; Dustman and Stickel, 1969; Stickel and Stickel, 1970). The toxicity of Kepone which we observed was similar to those reported previously by others with DDT. Coturnix in breeding condition exposed to DDT lived longer than birds not in breeding condition. Also males were more susceptible to DDT poisoning than females (Gish and Chura, 1970). The weight loss of cowbirds exposed to DDT which lived longer and which contained more fat at death than those dying earlier appeared directly related to the loss of fat (Stickel et al., 1970; Stickel and Stickel, 1970).
In the present report the Kepone-fed immature birds of both sexes probably had much less fat than the 27-week-old birds and the detoxification and elimination of Kepone were probably poorly developed. Decreased light could have caused a high mobilization of the fat causing intoxication and death. On the other hand, egg-laying has been shown to be a mechanism for ridding the body of residues of DDT and other chlorinated insecticides (Azavedo et al., 1965). It was possible that a higher fat content and better detoxification system in 27-week-old Kepone-fed birds of both sexes, in addition to egg-laying, probably was a factor in their improved viability. Under a 6L: 18D light cycle, it is doubtful that the decreased intake of Kepone ration was a factor in preventing mortality. ACKNOWLEDGMENT This study was supported by U. S. Public Health Service Grant ES 00054 for the Toxicology Center, and by Dr. R. A. Freedland’s laboratory. This investigation was presented in a more extended form as a thesis by the senior author in partial fulfillment of the requirements for the Ph.D. degree at the University of California, Davis, Cahfornia.
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7, 111-124.
GISH,C. D. ANDCHURA,N. J. (1970). Toxicity of DDT to Japanesequail as influencedby body weight, breedingcondition and sex. Toxicol. Appl. Pharmacol. 17, 740-751. HUBER,J. J. (1965). Somephysiologicaleffects of the insecticideKepone in the laboratory mouse.Toxicol. Appl. Pharmacol. 7,516-524. JACKSON, N., LINDSAY,R. K. ANDPEARCE, J. (1971).A study of the effectsof gonadalhormone administrationon someenzymesof amino-acidmetabolismin the oviduct of the immature domesticfowl. Comp. Biochem. Physiol. 39, 683-687. MCFARLAND,L. Z. ANDLACY, P. B. (1969). Physiologic and endocrinologic effects of the insecticideKepone in the Japanesequail. Toxicol. Appl. Pharmacol. 15, 44450. MATHER,F. B. AND WILSON,W. 0. (1964). Post-natal testicular developmentin Japanese quail (Coturnix cotarnix japonica). Poultry Sci. 43, 860-864. MEIER,A. H., MARTIN, D. D. AND MACGREGOR,R. (1971).Temporal synergismof corticosteroneand prolactin controlling gonadal growth in sparrows.Science 173, 1240-1242. NABER,E. C. ANDWARE,G. W. (1964). Effect of Kepone and Mirex on reproductive performancein the laying hen. Poultry Sci. 44, 875-880. OKA, T. ANDSCHIMKE, R. T. (1969a).Interaction of estrogenand progesteronein chick oviduct development.I. Antagonistic effect of progesteroneon estrogen-inducedproliferation and differentiation of tubular glandcells.J. Cell Biol. 41, 816-831. OKA, T. ANDSCHIMKE, R. T. (1969b).Interaction of estrogenand progesteronein chick oviduct development. II. Effects of estrogenand progesteroneon tubular gland cell function. J. Cell Biol. 43, 123-l 37. PALMITER, R. D. ANDWRENN, J. T. (1971).Interaction of estrogenand progesteronein chick oviduct development.III. Tubular gland cell cytodifferentiation. J. Cell Biol. 50, 598-615. SELYE, H. (1950). The Physiology and Pathology of Exposure to Stress. Acta. Inc., Montreal. SHERMAN, M. AND ROSS,E. (1961). Acute and subacutetoxicity of insecticidesto chicks. Toxicol. Appl. Pharmacol. 3,521-533. SILLER, W. G., DEWAR, W. A. AND WHITEHEAD, C. C. (1972).Cystic dilatation of the seminiferous tubulesin the fowl: A sequelof sodiumintoxication. J. Pathol. 107, 191-197.
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EFFECTS
IN QUAIL
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S. I., WEBER, W. C. AND REID, B. L. (1969). The effect of high levels of dietary DDT on egg production, mortality, fertility, hatchability and pesticide content of yolks in Japanese quail. PO&~ Sci. 48, 1000-1004. SMITH, S. I., WEBER, W. C. AND REID, B. L. (1970). Dietary pesticides and contamination of yolks and abdominal fat of laying hens. Poultry Sci. 49, 233-237. STICKEL, L. F. AND STICKEL, W. H. (1970). Distribution of DDT residues in tissues of birds in relation to mortality, body condition and time. In : Pesticides Symposium (W. P. Deichmann, ed.), pp. 57-65. Helios, Miami, Florida. STICKEL, W. H., STICKEL, L. F. AND COON, F. B. (1970). DDE and DDD residues correlated with mortality of experimental birds. In: Pesticides Symposium (W. P. Deichmann, ed.), pp. 287-294. Helios, Miami, Florida. TANAKA, K., MATHER, F. B., WILSON, W. 0. AND MCFARLAND, L. Z. (1965). Effect of photoperiods on early growth of gonads and on potency of gonadotropins of anterior pituitary in coturnix. Poultry Sci. 44, 662-665. VAN TIENHOVEN, A. (1961). Endocrinology of reproduction in birds. In: Sex and Internal Secretion (W. C. Young, ed.), Vol. 2, pp. 108881169. Williams & Wilkins, Baltimore, Maryland. WILSON, W. O., ABPLANALP, H. AND ARRINGTON, L. (1962). Sexual development of coturnix as affected by changes in photoperiods. Poultry Sci. 41, 17-22. WOODARD, A. E., ABPLANALP, H., WILSON, W. 0. AND VOHRA, P. N. (1965). Japanese quail husbandry in the laboratory. A. Woodard, Davis, California. SMITH,
AND
APPLIED
Photoperiods
PHARMACOLOGY
29,329-339
(1974)
and Age as Factors Modifying Kepone in Japanese Quail VICTORP. EROSCHENKO' AND
the Effects
of
WILBOR0. WILSON
Department of Anatomy, School of Veterinary Medicine, and Department of Avian Sciences, University of California, Davis, California 95616 Received August 14,1973; accepted March 21,1974
Photoperiods and Age as Factors Modifying the Effects of Kepone in JapaneseQuail. EROSCHENKO, V. P. AND WILSON, W. 0. (1974). Toxicol. Appl. Pharmacol. 29, 329-339.Immature Japanese quail (Coturnix coturnix japonica) of both sexesfed 200 ppm of the insecticideKepone, whenexposedto 16hr of light per day, hadsignificantly enlargedreproductive organs,livers and adrenalglands.After a reduction in the photoperiod high mortality occurred in only the birds previously fed Kepone. Administration of Keponeto adult femaleswhile egg-layingdid not affect the weight of reproductive organsor livers. In adult malesof the sameagetestesand livers weregreatly enlarged.In both sexesof adult birds, the adrenalswere enlarged.Subsequentexposureto reducedphotoperiod of the Kepone-fed adult quails of both sexesproduced no mortality. In malesfed Kepone, testicular regressionwasrecordedmuch earlier than in controls. Previous ingestionof Kepone affected neither the cessationof egg-layingnor the regressionof the ovary. Keponeexertedits effectsprimarily on the immature quail of both sexesand to a lesserdegreeon adults. An estrogenicaction of Kepone wasindicated by increasedoviduct weight of young femaleson a photoperiod of 6 hr light and 18hr dark per day. In adult malesthe major estrogeniceffect wasincreasedliver weight. Kepone’ (decachlorotetracyclodecanone) is a complex polycyclic chlorinated insecticide of high stability that has been found useful in controlling many speciesof insects,including roaches, ants, wireworm and leaf-eaters, aswell asfly larvae (Epstein and Legator, 1971; Brown, 1966). However, Kepone has been reported to interfere with the reproduction of mammals and birds and to affect various organs of the body. Various concentrations of Kepone in the diet reduced reproduction in mice (Huber, 1965), bobwhite quail, and pheasants (Dewitt et al., 1962, 1963) as well as decreased egg production in laying chickens (Naber and Ware, 1965).
Furthermore, Kepone has been shown to exert or promote “estrogenic” activity in the Japanese quail or common coturnix. Administration of Kepone to immature, hypophysectomized and ovariectomized quail resulted in an increase of the oviduct weight. Also, intact Kepone-fed quail exhibited enlarged ovaries characterized by a greater number of developing follicles, whereas Kepone-treated males had either
atrophic or greatly enlarged testes. An effect of Kepone that was common to both sexes of quail was an enlargement of the liver (McFarland and Lacy, 1969). 1Presentaddress:Departmentof BiologicalSciences, Universityof Idaho, Moscow,Idaho 83843. z Suppliedby Agricultural Divisionof Allied Chemicals, NewYork. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
329
330
EROSCHENKO
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The present study was undertaken to compare the weekly responses of various organs of the Japanese quail (Coturnix coturnix japonica) due to gonadotropins produced by photostimulation with the estrogenlike effects produced by feeding the insecticide Kepone. METHODS Immature and mature individuals of each sex from the University of California, Davis coturnix Random Line 908 were used in the experiments. The eggs were hatched in a forced-draft, single-stage incubator, and the chicks were brooded in commercial batteries with temperature and humidity regulated as described by Woodard et al. (1965) and Wilson et al. (1962). At 3 weeks of age the birds were separated according to sex based on plumage dimorphism, placed in individual wire-floor cages and exposed to 16 hr of light and 8 hr of darkness (16L : 8D) ; previously they were given continuous light (24L). The intensity of light was 30 foot-candles and was from an incandescent source. The ambient temperature of the room was kept at 22” rt 1“C. Birds of each sex were separated into control and experimental groups. The control group of birds was given turkey starter ration and water ad libitum. The experimental group was given the same diet except that 200 ppm of Kepone had been added. To ensure proper distribution, Kepone was first dissolved in acetone, thoroughly mixed into the feed, and then administered to the various experimental groups at the designated time. In experiments 1 and 3 the reduction of photoperiod occurred after Kepone administration. In experiment 2, the birds were allowed to mature under the reduced photoperiod prior to being fed Kepone (Fig. 1). After the reduction in the daily photoperiod the egg-laying performance of females from both the experimental and control groups of experiment 3 was recorded daily in order to establish the pattern of the cessation of egg laying. After egg laying had ceased groups of varying numbers of birds were sacrificed as scheduled. At the time of sacrifice in all the experiments, organs from both sexes of birds during both the stimulatory and inhibitory light regimens were excised and the weights of the ovary, oviduct, testes, liver, adrenal glands and carcass were taken on a Mettler balance and recorded. The oviduct was dissected, uncoiled and its length was measured to the nearest centimeter. All organs were fixed in formalin solution and prepared for subsequent histologic examination. The details of the designs for all 3 experiments are illustrated in Fig. 1. For statistical treatment of the data the Student t test was used to evaluate differences between means of the experimental and control group of the same age. A p value greater than 0.05 was not considered significant; however, when the probability for a given comparison was less than 0.05, these probabilities were listed. RESULTS Experiment
1
By the end of the first week of Kepone feeding a significant weight increase was recorded in the oviducts, adrenals and livers, whereas an increase in the weight of testes and ovaries was not significant until after the second and third week of Kepone feeding (Table 1). The reduction in light at 6 weeks of age from 16L : 8D to the 6L : 18D regimen had a severe effect on the survival rate of both sexes only when fed Kepone.
KEPONE EFFECTS IN QUAIL EXP.
331
I X
4
6 AGE
EXP.
10
8
(weeks)
2
AGEtweeks)
EXP.
3
x
0 E t E
--
x
x
x
X
t
6
2
II
24 AGE
26
2a
30
(weeks)
FIG. 1. Experimental design of three experiments with different photoperiodic treatment. The ages are on the abscissa: Key: X = Termination schedule of the quail during the experiments. K = Duration of feeding Kepone (200 ppm) to experimental group (both sexes) is shown within the arrows.
332
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WILSON
The immature males were most severely affected as 15 out of 21 died within the average time of 4.3 days, with a range from 1 to 9 days. Females were less severely affected; 10 of 17 died. The average time of death and range for the immature females was almost the same as for the males. The common characteristic in the dead birds of both sexes was a severe loss of total body weight. All experimental birds that were dying exhibited severe body tremors and ataxia. At the onset of body tremors there was no indication that birds consumed food. The result of postmortem examination indicated the birds that died earlier had heavier body weights. Birds that died at a later date were without visible body fat and had severely emaciated pectoral musculature. Kepone-fed birds of both sexes lost about 40-50 g of their total body weight within the mean time of 4.3 days prior to death. Postmortem examination of various organs indicated that the mean liver, testes, ovarian and oviducal weight of the dead birds was significantly less than the corresponding mean organ weight from the birds terminated just prior to light reduction. TABLE 1 EFFECT LENGTH
OF INSECTICIDE KEPONE OR WEIGHT OF ORGANS
Parameters measured Kepone treatment duration (weeks) Oviduct length (cm) Control Kepone fed Oviduct wt (g) Control Kepone fed Ovarian wt (g) Control Kepone fed Testes wt (g) Control Kepone fed Adrenal wt (mg) Control Kepone fed Control Kepone fed Liver wt (g) Control Kepone fed Control Kepone fed a (0) = Number b p < 0.05. cp < 0.01.
dp < 0.001.
of birds.
OF 16L: SD ON THE MEAN OF BOTH SEXES IN EXPERIMENT 1
(200 PPM) AND A PHOTOPERIOD IN YOUNG
Sex
COTURNIX
3 0
Age of birds (weeks) --____-~~~~ 5 -
1
6
__~---
2
3 13.3 (10) 24.2 (7)
F F
3.6 (7) -
3.7 (7) 8.4 (7)d
6.5 (8) 14.8 (7)d
F F
0.02 (7) -
0.03 (7) 0.68 (7)d
0.45(8) 2.02 (7)d
2.57 (10) 4.96(7)b
F F
0.04(7) -
0.05(7) 0.08 (7)
0.09(8) 0.17(7)
6.82 (7)d
M M
0.10 (7) -
0.21 (7) 0.34(7)
0.49 (7) 1.57 (7)
1.59(7) 2.72 (8)"
F F M
8.60(7)
9.24(7) 10.71 (7)b 9.66(7) 12.56(7)b
10.39(8) 14.69(7) 12.12 (7) 15.29(7)
12.81 (10)
M
7.2;(7) -
F F M M
2.72(7) 2.37 (7) -
3.60(7) 4.93 (7) 3.02 (7) 4.33 (7)d
3.19(8) 6.63 (7)d 3.19 (7) 4.51(7)
1.44 (10)
18.36 (7) 11.07 (7) 17.36 ( 8)d
3.90 6.74 2.63 7.75
(10)
(7)d (7) (8)d
KEPONE
EFFECTS
333
IN QUAIL
On the other hand, the mean weight of the adrenal glands from both sexes of birds that died remained unchanged. The birds of both sexes that survived the initial 7 to 9 days of light reduction did not show body tremors, continued to eat food and appeared normal. The surviving birds were observed daily for an additional 4 weeks after the light change and were terminated when 10 weeks of age. None of the control birds died after the light was reduced. Experiment 2
After 1 week of the Kepone-containing diet significant weight changes were observed in the oviducts and livers, whereas the weight of adrenal glands did not change significantly until after the second and third week of Kepone feeding (Table 2). After 3 weeks of treatment with Kepone, the ovarian weights were 37 and 30 mg and testicular weights were 16 and 13 mg for the experimental and control groups, respectively. Thus under reduced photoperiods the gonads were not significantly altered during the entire 3 weeks of Kepone treatment. No observable body tremors, ataxia or death were recorded in any Kepone-treated or control birds of either sex. TABLE 2 EFFECT
OF INSECTICIDE KEPONE (200 ppm) AND A PHOTOPERIOD LENGTH AND WEIGHT OF ORGANS IN COTURNIX OF BETH
OF 6L: 18D ON THE MEAN SEXES IN EXPERIMENT 2
Age of birds (weeks) Parameters measured Kepone treatment duration (weeks) Oviduct length (cm) Control Kepone fed Oviduct wt (g) Control Kepone fed Liver wt (g) Control Kepone fed Control Kepone fed Adrenal wt (mg) Control Kepone fed Control Kepone fed
Sex
6
7
8
9
0
1
2
3
F F
3.4 (7) -
3.6 (7) 9.2 (7)
3.6 (7) 12.7 (7)
3.9 (7) 15.8 (7)
F F
0.02 (7) -
0.02 (6) 0.72 (7)
0.02 (7) 1.19 (7)
0.03 (7) 1.60 (7)
F F M M
2.72 (7)
2.94 (7) 4.69 (7)’ 2.65 (8) 3.67 (7)*
3.01 (7) 6.25 (7) 2.62 (7) 4.44 (7)”
2.84 (7) 6.41 (7) 2.27 (7) 5.95 (7)
F F M M
10.60 (7)
9.70 (7) 10.80 (7) 9.91 (7) 12.60 (7)b
9.30 (7) 14.50 (7) 10.40 (7) 12.40 (7)b
3.10 (8) -
l&8) -
11.20 (7) 12.20 (7) 8.50 (8) 9.62 (7)
a (0) = Number of birds. *p < 0.01. ep < 0.001.
Experiment 3
Neither 3 weeks of Kepone administration to egg-laying quail nor 2 weeks of reduced photoperiod (6L: 1SD) produced any significant changes in the mean oviduct length,
334
EROSCHENKO
AND
WILSON
nor in the oviduct, ovarian, and liver weight. But, after 3 weeks of reduced light significant changes were recorded in the mean oviduct length, oviduct and liver weight. The ovaries were completely unaffected during the Kepone feeding or after the reduction of photoperiod (Table 3), whereas the adrenal glands were significantly heavier only at the end of 3 weeks of Kepone feeding. Daily observation, after reduction of light, demonstrated that the average time required for cessation of egg-laying in both the control and experimental birds was 8.0 and 8.3 days, respectively, and was consequently statistically insignificant. Three weeks of Kepone feeding to adult males resulted in significant weight increases in the adrenal gland, liver and testes. Reduction of the photoperiod for 1 week did not TABLE EFFECT
OF INSECTICIDE MEAN WEIGHT
3
KEPONE (200 ppm) DURING REDUCED PHOTOPERIODS ON LENGTH OF ORGANS IN SEXUALLY MATURE COTURNIX IN EXPERIMENT 3
AND
Age of birds (weeks) Parametersmeasured Sex
27
28
29
30
31
Length without Kepone (weeks) after reduction of light to 6L: 18D:
0
1
2
3
4
F
25.6 (7) 26.5 (7)
Laying Laying
10.5 (11) 10.5 (11)
8.3 (12) 9.7 (1l)b
7.3 (9) 8.3 (9)
F F
6.37 (7) 6.29 (7)
Laying Laying
0.74 (11) 0.82 (11)
0.37 (12) 0.51 (1l)b
0.27 (9) 0.34 (9)
F F
6.21, (7) 5.45 (7)
Laying Laying
0.12 (11) 0.12 (11)
0.10 (12) 0.11 (11)
0.07 (9) 0.09 (10)
M M
2.73 (7) 3.86 (7)
2.28 (12) 1.06 (12)”
0.39 (12) 0.31 (9)
0.09 (10) 0.02 (12)b
0.05 (7) 0.03 (13)
ll.oo(ll) 12.20(11) 10.61(12) 13.31(1l)b
Oviduct length (cm) Control Kepone fed Oviduct wt (g) Control Kepone fed Ovarian wt (g) Control ‘Keponefed Testeswt (g) Control Kepone fed Adrenal wt (mg) Control Keponefed Control Kepone fed Liver wt (g) Control Kepone fed Control Kepone fed
F
F F M M
12.91 16.50 11.52 15.30
(7) (7)b (7) (7)b
Laying Laying 11.30(12) 12.90(12)
F F M M
6.34 6.88 2.59 5.35
(7) (7) (7) (7)d
Laying Laying 2.84 (12) 6.24 (12)d
1 Ly)O=OJumber of b’lr d s’, 1aying females were not sampled. =p < 0:01: dp < 0.01.
3.71 3.67 3.18 5.72
(11) (11) (12) (9)d
12.00 13.31 11.42 13.91
(12) (11) (10) (12)b
11.40 11.90 12.01 12.13
(9) (9) (7) (13)
3.42 4.24 3.29 6.28
(12) (1 l)b (10) (12)b
3.42 4.40 2.62 4.29
(9) (10)b (7) (1 3)d
KEPONE
EFFECTS
IN QUAIL
335
affectthemeantesticularweightin thecontrolbirds.However,thetestesfromKeponefed birds were significantly smaller than those from the control birds after 1 and 3 weeksof reducedphotoperiod. The meanliver weight from experimental birds remained significantly heavier during the entire 4 weeks of reduced light, whereas the adrenal glands were significantly heavier during various intervals after the light reduction (Table 3). The reduction of light at 27 weeks of age did not produce any body tremors, ataxia, visible ill-effects or mortality in either the Kepone-fed or control group birds of either sex. DISCUSSION Increases in both weight and length of the oviduct in the immature control birds under stimulatory light was initially recorded after the 4th week of age. However, the oviducts in the immature quail subjected to a reduced light cycle of 6L : 18D remained undeveloped, A light regimen of 16L:8D was shown to be stimulatory to gonadal development of young chicks of either sex. Under these light conditions females produced eggs at 7 to 8 weeks of age and spermatogenic activity was see%in males at about 7 weeks of age. Decrease in daylight from 18 to 6 hr delayed or inhibited sexual maturation and was nonstimulatory to the gonads (Wilson et al., 1962; Mather and Wilson, 1964; Follett and Farner, 1966; Tanaka et al., 1965). Reports indicate that the oviduct of immature avian species differentiates rapidly when subjected to exogenous hormones. Exogenous estrogenic compounds administered to immature chicks produced an increase in the oviduct weight and development of tubular glands (Oka and Schimke, 1969a, b; Palmiter and Wrenn, 1971; Jackson et al., 1971). Certain insecticides exert estrogenic effect on the reproductive organs of birds. Treatment of immature chicks and Japanese quail with o,p’-DDT produced enlarged oviducts and increased glycogen content of the oviducts (Bitman et al., 1968; Cooke, 1970). Administration of Kepone to sexually immature chickens likewise produced enlarged oviducts (Sherman and Ross, 1961). The insecticide Kepone exerted a highly stimulatory effect on the oviducts of sexually immature quail. Accelerated growth in length and weight of the oviduct was recorded within the first 2 weeks of Kepone administration. The most pronounced effect was noted in the oviducts of immature quail subjected to reduced (6L : 18D) light regimen. The oviducts were enlarged more than 60 times over the controls. Histology of the oviducts in the regions of the magnum, isthmus and uterus showed cellular hypertrophy, hyperplasia, ciliated surface epithelium, formation of tubular glands and accumulation of secretory material in these cells (submitted for publication). In laying quail, on the other hand, Kepone ration did not alter the weight or the length of the oviduct. Histologic examination of these oviducts did not indicate any detectable cellular alterations. Similar results were obtained when Kepone ration was given to female quail just beginning to lay eggs. Thus it appears that Kepone exerted an “estrogenlike” effect on the undeveloped oviducts of only maturing quail, whereas no additional effect was seen in the fully functional oviduct of the laying quail. In experiments with ovariectomized and hypophysectomized quail, McFarland and
336
EROSCHENKOAND WILSON
Lacy (1969) postulated that Kepone could stimulate the hypothalamic-pituitary axis to release: FSH and thereby stimulate ovarian estrogen release as well as direct stimulation of the immature oviduct in the absence of the pituitary. The greatest estrogenlike effect of Kepone occurred in quail with the intact pituitary. Kepone produced changes neither in the ovarian weight nor in egg production in the laying quail. Furthermore, Kepone did not affect the developing ovary in the maturing quail exposed to stimulatory light until after the fifth week of age. The ovaries were characterized by 3 to 4 larger follicles and a greater number of smaller developing follicles. McFarland and Lacy (1969) reported a reduced number of eggs in the uterus of Kepone-fed quail, thus implying a reduced egg production. In Kepone-treated adult mice, normal follicular development with few corpora lutea was recorded, Hormone bioassay showed that the mice were under prolonged stimulation of FSH and estrogen but under insufficient LH stimulation. This blocking of rhythmic release of LH by Kepone could prevent ovulation and corpora lutea formation in mice (Huber, 1965). The effect of Kepone on the ovaries of maturing quail exposed to stimulatory light could be similar to that reported in mice by Huber (1965), whereas its effect on the quail oviduct could have a dual action as postulated by McFarland and Lacy (1969). The increase inpviduct size, weight and cellular differentiation that we found during the reduced light (6L: 18D) and the absence of increased ovarian weight strongly suggests a direct estrogenic action of Kepone on the oviduct. Quail of both sexes fed Kepone had enlarged adrenals. The heaviest adrenals were recorded in the birds that were undergoing sexual maturation under stimulatory light. Due to such increases in adrenal gland size in all Kepone-fed birds, it was highly probable that the action of Kepone was similar to that of an internal stressor and the response of the adrenals followed the classical concept of changes associated with stress (Selye, 1950). Kepone administration to maturing and adult males under a 16L:8D light cycle produced heavier testes. The testes appeared edematous and histologic examination revealed a great distention of seminiferous tubules, apparently containing fluid (submitted for publication). Also Kepone appeared to exert its estrogenlike action on the testes by reducing their weight below that of controls after the first week of light reduction. It has been reported that physiologic doses of estrogen or prolactin reduces weight of enlarged testes, depresses spermatogenesis and inhibits gonadal growth (Van Tienhoven, 1961; Meier et al., 197 1). It has been also shown that testes enlargement occurred in unstressed chicks after deoxycorticosterone acetate (DCA), cortisone and ACTH administration (Conner, 1959). Histologic appearance of testes from DCAtreated chicks showed a dilated seminiferous tubule system, and a stretched and flattened germinal epithelium (Boas, 1958). Furthermore, high salt diet given to immature chicks produced hypertrophy of the seminiferous tubules and later transformed them into thin-walled cysts containing water-clear fluid (Siller et al., 1972). The insecticide Kepone likewise produced increased testicular weight and an associated distention of seminiferous tubules by accumulation of waterlike fluid. An important observation in this study was the fact that a reduction of light to 6 hr per 24 hr at 6 weeks of age produced a very high mortality rate in the Kepone-fed quail of both sexes. Young males were affected more than the females although the mean time of death and the signs of intoxication were similar to those reported in mice
KEPONE EFFECTSIN QUAIL
331
(Huber, 1965), in chickens (Sherman and Ross, 1961), in pheasants (Azavedo et al., 1965) and in cowbirds (Stickel etal., 1970). Decreased weights of body carcass, reproductive organs, absence of visible fat stores and emaciation were the striking features observed in the birds that died. Similar light reduction failed to produce either the signs of intoxication or mortality in Kepone-fed adult quail of either sex. Also, feeding Kepone during the reduced light to 6-week-old birds likewise did not cause mortality. Since death in the younger birds of both sexes occurred after prolonged tremors and since there was a high weight loss, it was possible that available fat stores were mobilized at this time. Chlorinated hydrocarbon pesticides are fat soluble and have been shown to accumulate in fatty tissue of hens and in the egg yolk (Smith et al., 1970). In laying hens fed different concentrations of Kepone, egg yolk residues of Kepone reached a maximum by the fifth week (Naber and Ware, 1964). Other chlorinated pesticides such as DDT were shown to accumulate in egg yolk of chickens and Japanese quail in proportion to dietary levels of DDT (Smith et al., 1969, 1970). Furthermore, 10% or more of the daily intake of DDT in pheasants can be excreted by laying females in their eggs, thus reducing the quantity of ingested DDT placed into other fat deposits (Azavedo et al., 1965). Since the chlorinated insecticides are stored in the body fat, the mobilization of this stored insecticide would have an effect on the well being of the bird. Mortality is most likely to occur at times of seasonal or emergency weight loss and during other stresses, for then the stored toxicant is released into blood and other tissues causing toxicity (Ecobichon and Saschenbrecker, 1968; Dustman and Stickel, 1969; Stickel and Stickel, 1970). The toxicity of Kepone which we observed was similar to those reported previously by others with DDT. Coturnix in breeding condition exposed to DDT lived longer than birds not in breeding condition. Also males were more susceptible to DDT poisoning than females (Gish and Chura, 1970). The weight loss of cowbirds exposed to DDT which lived longer and which contained more fat at death than those dying earlier appeared directly related to the loss of fat (Stickel et al., 1970; Stickel and Stickel, 1970).
In the present report the Kepone-fed immature birds of both sexes probably had much less fat than the 27-week-old birds and the detoxification and elimination of Kepone were probably poorly developed. Decreased light could have caused a high mobilization of the fat causing intoxication and death. On the other hand, egg-laying has been shown to be a mechanism for ridding the body of residues of DDT and other chlorinated insecticides (Azavedo et al., 1965). It was possible that a higher fat content and better detoxification system in 27-week-old Kepone-fed birds of both sexes, in addition to egg-laying, probably was a factor in their improved viability. Under a 6L: 18D light cycle, it is doubtful that the decreased intake of Kepone ration was a factor in preventing mortality. ACKNOWLEDGMENT This study was supported by U. S. Public Health Service Grant ES 00054 for the Toxicology Center, and by Dr. R. A. Freedland’s laboratory. This investigation was presented in a more extended form as a thesis by the senior author in partial fulfillment of the requirements for the Ph.D. degree at the University of California, Davis, Cahfornia.
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AND
WILSON
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