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Response of the Reproductive Organs of the Japanese Quail to Pinealectomy and Melatonin Injections1
314
W. A. BECKER, J. V. SPENCER AND J. L. SWARTHOOD
wood, 1964. The pre-incubation storage of turkey eggs in closed environments. Poultry Sci. 43: 1526-1534. Cotterill, O. J., R. A. Gardner, E. M. Funk and F. E. Cunningham, 1958. Relationship between temperature and carbon dioxide loss from shell eggs. Poultry Sci. 37: 479-483. Funk, E. M., and J. Foward, 1960. Effect of holding temperature on hatchability of chicken eggs. Missouri Agr. Exp. Sta. Bull. 732. Krueger, W. F., W. E. McCune, C. B. Ryan, R. C. Jaska and J. H. Quisenberry, 1962. Optimum temperature and humidity for holding hatching eggs for short periods. Texas Agr. Exp. Sta. M. P. 592.
Landauer, W., 1961. The hatchability of chicken eggs as influenced by environment and heredity. University of Connecticut Monograph 1. McDonald, M. W., 1960. Effect of temperature of storage and age of fowl eggs on hatchability and sex ratio, growth, and viability of the chickens. Austral. J. Agr. Res. 11: 664-672. Proudfoot, F. G., 1964. The effects of plastic packaging and other treatments on hatching eggs. Canadian J. Anim. Sci. 44: 87-95. Proudfoot, F. G., 1965. The effect of film permeability and concentration of nitrogen, oxygen and helium gases on hatching eggs stored in polyethylene and Cryovac bags. Poultry Sci. 44: 636-644.
Response of the Reproductive Organs of the Japanese Quail to Pinealectomy and Melatonin Injections1 KAZUTAKA HOMMA2, LARRY Z. MCFARLAND AND WILBOR 0. WILSON Departments of Poultry Husbandry and. Anatomy, University of California, Davis, California 95616 (Received for publication May 12, 1966)
I
N recent years research on mammalian pineal physiology has been active. Melatonin elaborated by the pineal gland inhibits gonadal activity in rats (Wurtman et al, 1963a; Chu et al, 1964). The synthesis of melatonin in the rat pineal is controlled through environmental photoperioids, viz. it is accelerated by darkness and inhibited by light (Wurtman et al., 1963b). These diurnal changes in melatonin content of the pineal have been called the melatonin rhythm (Quay, 1964) Serotonin, the precursor of melatonin, also undergoes a marked diurnal change in the pineal (Quay, 1963), but there is no strict reciprocal correlation between the two rhythms. When rats were placed in continuous darkness or blinded, the melatonin 'Supported by research grant NB-04171 from National Institute of Neurological Diseases and Blindness. 2 Department of Animal Physiology, Faculty of Agriculture, Nagoya University, Anzyo, AichiKen, Japan.
rhythm was soon abolished, but the serotonin rhythm was maintained for more than 2 weeks, suggesting that the melatonin rhythm was under an exogenous control, whereas, serotonin rhythm was under an endogenous control (Snyder et al., 1964). Melatonin seems to play a role in controlling gonadal activity, since low doses of melatonin are more effective than serotonin in inhibiting ovarian activity of rats subjected to continuous light (Chu et al., 1964). Melatonin pharmacologically depresses the activity of the intestinal and uterine muscles with 300 [AM/ml. of melatonin completely inhibiting the spontaneous movements of rat uterine muscle in vitro (Hetrz-Eschel and Rahamimoff, 1965). Melatonin also acts as an antagonist toward certain types of chemical and auditory stimuli in the central nervous system ( Arutyunyan et al., 1964). Experiments on pineal physiology in avian species are not entirely in agreement with the results obtained in mammals. Con-
PINEALECTOMY AND MELATONIN INJECTIONS
trary to the results in rats, Axelrod et al., (1964) reported that the pineal enzyme HIOMT (hydroxyindole-O-methyl transferease), an enzyme necessary for melatonin synthesis, increased in activity as did the pineal weight in chickens placed in constant light. Chickens kept under diurnal photoperiods had higher HIOMT activity during the light period in contrast to that during the dark period. The high activities of this enzyme in an avian species (Quay, 1965) suggested the possibility that birds can produce larger amounts of melatonin than mammals. Furthermore, photoperiods favoring melatonin production are known to stimulate gonadal development in the chicken (Axelrod et al., 1964). The present experiments were designed to find out whether the pineal gland and melatonin are involved in the mechanism of photocontrol of the avian gonads. The Japanese quail (Coturnix coturnix japonica) was selected since gonad development of this species is known to be sensitive to environmental photoperiods (Wilson et al., 1962; Tanakae^aZ., 1965). MATERIALS AND METHODS
The source of Japanese quail and their general care were the same as reported previously (Tanaka et al., 1965). Pinealectomy. Two different procedures for pinealectomy were used depending upon the age of the birds. For quail younger than 3 weeks of age, high frequency electrocautery was used to destroy the pineal directly through the skull. This was feasible at this age, since ossification of the temporal bone was not advanced and the pineal body lay adjacent to the skull. After 4 weeks of age this procedure of pinealectomy was no longer applicable due to advanced ossification and the migration of the pineal deep between the cerebral hemispheres and cerebellum. In these birds the
315
bone was carefully removed using the dental drill and the pineal gland was surgically removed with its stalk. Local application of thrombin was used to minimize hemorrhage. The success of each pinealectomy was microscopically verified at autopsy. Only data from completely pinealectomized quail without brain damage were included in the analysis. The experiment involving length of photoperiod and pinealectomy comprised of a series of tests done at different seasons of the year and involving of separate environmental facilities. For this reason each group has its own control and the statistical analyses involved the t test rather than a more sophisticated statistical analysis. The effects of pinealectomy on gonadal growth were investigated in young quail of both sexes under 4 different photoperiods. Two were stimulatory photoperiods: continuous light (24 LL) and repeated cycles of 14 hours of light: 10 hours of dark (14L:10D). The other two light regimens were non-stimulatory (12L:12D, and 8L:16D), in that these photoperiods delayed gonadal growth for several weeks after hatch. Twenty to 36 quail were exposed to each photoperiod, approximately half of the quail from each photoperiod group were pinealectomized at 1 week of age, and the other half served as controls. All were killed at 4 weeks of age, and the gonads removed and weighed to the nearest 0.1 mg. In another experiment, quail were exposed to an 8L:16D photoperiod from hatch to 7 weeks of age. At 4 weeks of age, 13 males and 9 females were surgically pinealectomized, 17 males and 7 females were sham-operated, and 10 males and 7 females were used as non-operated controls. These birds were autopsied at 7 weeks of age. Melatonin treatments. In an experiment with young quail, the effect of exogenous melatonin was investigated by implanting
316
K. HOMMA, L. Z. MCFARLAND AND W. O. WILSON TABLE 1.—The results of four separate~tests designed to study the effects of pinealectomy at 1 week of age on body and gonadal weights of Japanese quail under four different photoperiods. Means + Standard error (number of samples) Photoperiods Body wt. at 4 wks. (g.)
Group
Testes wt. at 4 wks. (mg.)
Ovary wt. at 4 wks. (mg.)
Oviduct wt. at 4 wks. (mg.) 6 8 . 6 ± 1 0 . 2 (11) 6 3 . 2 + 1 1 . 0 (11)
Pinealectomy Control
24 L: O D 24 L: O D
92.6 + 1.4 92.5 + 2.2
654.3 + 65.2 (18) 599.5 + 74.5 (IS)
8 8 . 6 + 1 0 . 4 (19) 75.8+ 3.8 (17)
Pinealectomy Control
14 L: 10 D 14 L: 10 D
87.5 + 1.3*: 83.3 + 1.1
507.0 + 86.0 (11) 600.5 + 49.1 (13)
91.2 + 19.5 (9) 293.7±96.4 (9)* 65.6 + 5.9 (12) 121.9 + 38.9 (12)
Pinealectomy Control
12 L : 1 2 D 12 L:12 D
84.5 + 1.2 85.3 + 1.5
14.8 + 0.7 (8) 14.5 + 1.0 (12)
35.1 + 3.8 (6) 3 5 . 4 ± 3 . 6 (8)
17.3 + 1.4 (6) 15.5 + 0.8 (8)
Pinealectomy Control
8 L : 16 D 8L:16D
84.2 + 2.0*: 79.0 + 0.5
10.8 + 0.6 (12) 9 . 3 + 0 . 5 (13)
2 9 . 3 ± 3 . 2 (6) 22.6 + 2.1 (5)
17.4 + 0.5 (6) 16.7 + 1.5 (5)
: Means of body weights include both sexes. Means significantly (P<0.05) different from the control of the same photoperiod.
melatonin pellets. One hundred and twenty-six quail were divided into 4 groups soon after hatch. Each group was implanted at one week of age with small cylindrical 25 mg. pellets of cholesterol to which was added either 0, 1, 10, or 100 u,g. of melatonin. The average body weight of the quail at 1 week was approximately 18 g. The pellets were implanted subcutaneously on the dorsal aspect of the neck, after which time all birds were raised together under continuous light. They were killed at 4 weeks of age, at which time all the implanted birds still had a remnant of the pellet at the site of implantation. The effects of melatonin on the oviposition time were investigated in 10 laying quail. They were exposed to a 16L:8D photoperiod for 32 days. A record of egg production and time of lay was taken throughout this period. Every other day a TABLE 2.-
dye was administered orally (0.05 ml. of a Sudan III solution in corn oil) to each hen. Eggs were collected daily, boiled and sliced. Colored rings in the yolk were used as indices to estimate the number of maturing ovarian follicles. On day 21, 22, 23, and 24, these quail were injected subcutaneously with 1.0 mg. of melatonin dissolved in propylene glycol, and the record of egg production after melatonin treatment was compared with that preceding treatment. RESULTS AND DISCUSSION Pinealectomy. The gonadal and oviducal weights of 4 week old pinealectomized and control quail are shown in Table 1. Since the experiments involving various photoperiods shown in Table 1 are conducted in different seasons of the year and in different environmental facilities, considerable deviation can be expected from one
-Effects of pinealectomy at 4 weeks of age on body and gonadal weight of Japanese quail under 8 L: 16 D photoperiod. Means + Standard errors (number of samples) Body wt. (g)
Group Pinealectomy Sham operation Control
Male
Female
104.8 + 1.7(15) 104.8+1.4(13) 102.0 + 2.2(10)
104.5 + 1.6(9) 104.8 + 2.0(9) 99.0 + 2.5(7)
Testes wt. Ovary wt. Oviduct wt. at 7 wks. (mg.) at 7 wks. (mg.) at 7 wks. (mg.) 27.5 + 1.3(15) 38.9 + 4.6(9) 33.0 + 5.6(12) 41.6 + 3.0(9) 36.6+12.5(10) 45.7 + 6.1(7)
* Means are significantly (P<0.05) different from control.
20.1+2.2(9)* 19.2 + 1.5(9)* 30.4 + 5.2(7)
PINEALECTOMY AND MELATONIN INJECTIONS
317
TABLE 3.—Effects of 1,10 and 100 ng. melatonin implants made at 1 week of age on body and gonadal weight in young Japanese quail under continuous light. Means + Standard errors {number of samples) Body wt. at 4 wks. (g.) Group Male Control leg 10 ng 100 Mg
90.5 + 1.3(11) 87.1 + 5.1(22) 85.8 + 2.1(12) 83.4+1.2(18)
Female 87.1 + 82.7 + 89.3 + 86.4 +
Testes wt. at 4 wks. (mg.)
5.1(11) 5 8 8 . 6 1 2 9 . 7 " ^ , 2.2(15) 279.9 + 4 9 . 8 * ^ a 2.9(21) 400.5 + 80.4 ~ - > a 2.0(16) 598.4 + 7 4 . 5 ^
Ovary wt. at 4 wks. (rag.)
Oviduct wt. at 4 wks. (mg.)
89.1+6.7 65.5 + 5.8* 76.5±6.8 81.2 + 7.8
82.7 + 12.3 46.1 + 6.0* 83.8+17.5 68.3 + 11.7
' Means are significantly (P<0.05) different from control. Means are significantly different ( P < 0 . 0 5 ) .
test to another; thus, it was thought desirable to analyze the data by a separate t test for each photoperiod rather than by analysis of variance of the whole data. Under the three types of photoperiods, i.e., 24 LL, 12L:12D, and 8L:16D, pinealectomized quail showed no statistically significant difference either in gonadal or oviducal weight from that of the controls. Under 14L:10D photoperiod, pinealectomized females showed a statistically significant increase in oviducal weight ( p < 0.05), but the slight increase in ovarian weight in the pinealectomized birds was not significant. The gonadal development of quail operated on at 4 weeks and killed at 7 weeks of age is shown in Table 2. In this experiment, both pinealectomy and sham operation caused a comparative decrease in gonadal weight (not significantly different from control), and in oviducal weight (significantly different from control, p < 0.05). Although there was no sign of retardation of body growth in the two operated groups, the results suggest that retardation of oviducal growth in the pinealectomized birds was induced by surgical stress and not by removal of the pineal gland.
tardation of gonadal and oviducal growth (p < 0.05). However, with increasing amounts of melatonin the gonadal inhibition was decreased rather than increased. At the highest dose of 100 \i.g., melatonin apparently had no gonadal inhibitory effect. This type of dose-response relationship can be expected only when gonadal inhibitory effect of melatonin is indirect and could offer an explanation for the high HIOMT activity of the pineal gland in sexually active birds of various species (Quay, 1965). The results of melatonin injection into laying quail are shown in Figure 1. The time of injection is indicated by arrow (1700 hours). The distribution of the time of lay was based on number of eggs collected during a 22 day period prior to the melatonin injection (170 eggs), and during a 10 day period following melatonin injection (95 eggs). Melatonin injection significantly (p <0.01) affected the time of lay (Chi-square test, p < 0 . 0 1 ) . The major peak of lay at 2100 hours prior to the treatment was advanced 2 hours following melatonin. Furthermore, some new minor peaks appeared after melatonin treatment. A daily dose of 1 mg. did not affect the number of developing follicles in the ovary (Table 4), and no changes in the Melatonin implantation. The effects of behavior of the birds were observed. exogenous melatonin on gonadal weight in The results of the present experiments young quail under continuous light are favor the view that the pineal is involved in shown in Table 3. Quail implanted with the mechanisms regulating gonadal func1 jtg. of melatonin showed a significant re- tion in the quail, presumably through se-
31S
K. HOMMA, L. Z. MCFARLAND AND W. O. WILSON
^o PRIOR TO TREATMENT FOLLOWING TREATMENT
<
S20 < O
0
2
4
6
8
10 12 14 16 TIME IN HOURS
18 2 0 22 24
LIGHT OFF
LIGHT ON
FIG. 1. Distribution of eggs laid by quail before and after melatonin injections. Time is expressed on 24 hour clock.
cretion of melatonin. Undoubtedly, the pineal is related to certain pathways which transmit environmental stimuli to the pituitary, but the participation of the pineal is not strong enough to modify the entire gonadal response to various environmental stimuli, i.e., pinealectomy did not restore the gonadal growth of those quail raised under non-stimulatory photoperiods. One of the important roles of the pineal would be that of a monitor which prevents hypersecretion of pituitary hormones (Moszkowska, 1965), or that of "Zeitgeber" or
timing device which regulates the time of hormone release as was indicated in the experiments of laying quail (Figure 1). Shellabarger reported that pinealectomy in the chicken caused gonadal hypertrophy in old cockerels (1952) and gonadal inhibition in young cockerels (1953). It appears from his results that the function of the pineal is influenced by age as well as by environmental factors. Such an age effect was not evident in quail under a non-stimulatory photoperiod (8L:16D). The mechanism whereby melatonin causes gonadal inhibition in mammals is still not fully understood. A direct inhibition of ovarian activity has been proposed, based on the evidence that labeled melatonin was accumulated by the rat ovary (Wurtman et al., 1964). This would not appear to be the case in quail, since higher doses of melatonin were not more inhibitory (Table 3). SUMMARY Pinealectomy of young coturnix quail permitted rapid oviducal growth in the female under 14L:10D photoperiod, but was without effect in quail kept under nonstimulatory photoperiods. Melatonin implantation in 1 week old birds at a level of 1 u.g. per bird inhibited gonadal growth, while higher levels were less effective. Subcutaneous injection of 1 mg. of melatonin for 4 days changes the distribution of the time of lay, but did not inhibit ovarian activity. REFERENCES
TABLE 4.—Number of developing follicles in the ovary before and after melatonin injection 1 mg.j'day/bird for 4 days)
Before injection After injection
N
Number of observation
«™£* of nj^Ajev (Mean+S.E.).
36 30
3.4 + 0.3 3.3 + 0.3
Arutyunyan, G. S., M. D. Mashkovskii and L. F. Roshchina, 1964. Pharmacology of melatonin. Fed. Proc. 23 :T. 1330-1332. Axelrod, J., R. J. Wurtman and C. W. Winget, 1964. Melatonin synthesis in the hen pineal gland and its control by light. Nature, 201: 1134. Chu, E. W., R. J. Wurtman and J. Axelrod, 1964. An inhibitory effect of melatonin on the es-
PlNEALECTOMY AND MELATONIN INJECTIONS trous phase of the estrus cycle in rodents. Endocrinology, 75: 238-242. Hertz-Eschel, M., and R. Rahamimoff, 1965. Effects of melatonin on uterine contractivity. LifeSci. 4 : 1367-1372. Moszkowska, A., 1965. Contribution a l'etude du mecanisme de 1'antagonisme epiphyso-hypophysaire. In Structure and Function of the Epiphysis Cerebri. Ed. Aliens Kappers, J., and J. P. Schade. pp. 564-576, Elsevier Pub. Co., Amsterdam. Quay, W. A., 1963. Circadian rhythm in rat pineal serotonin and its modification by estrous cycle and photoperiod. Gen. Comp. Endocr. 3 : 473-479. Quay, W. A., 1964. Circadian and estrous rhythms in pineal melatonin and 5-hydroxyindole-3-acetic acid. Proc. Soc. Exp. Biol. Med. 115: 710-713. Quay, W. A., 1965. Retinal and pineal hydroxyindole-O-methyl transferase. Life Sci. 4 : 983991. Shellabarger, C. J., 1952. Pinealectomy vs. pineal injection in the young cockerel. Endocrinology, 5 1 : 151-154. Shellabarger, C. J., 1953. Observation of the pi-
319
neal in the White Leghorn capon and cockerel. Poultry Sci. 32 : 189-197. Snyder, S. H., M. Zweig and J. Axelrod, 1964. Control of the circadian rhythm in serotonin content of the rat pineal gland. Life Sci. 3 : 1175-1179. Tanaka, K., F. B. Mather, W. 0. Wilson and L. Z. McFarland, 1965. Effect of photoperiods on early growth of gonads and on potency of gonadotropins of the anterior pituitary in coturnix. Poultry Sci. 44: 662-665. Wilson, W. O., H. Abplanalp and L. Arrington, 1962. Sexual development of coturnix as abfected by changes in photoperiod. British Poultry Sci. 3 : 105-113. Wurtman, R. J., J. Axelrod and E. Chu, 1963a. Melatonin, a pineal substance: Effect on the rat ovary. Science, 141: 277-278. Wurtman, R. J., J. Axelrod and L. S. Phillips, 1963b. Melatonin synthesis in the pineal gland controlled by light. Science, 142: 1071-1073. Wurtman, R. J., J. Axelrod and L. R. Potter, 1964. The uptake of H3-melatonin in endocrine and nervous tissues and the effects of constant light exposure. J. Pharmacol. Exp. Ther. 143:314-318.
Studies of Dietary Piperazine, Phenothiazme, and Dibutyltin Dilaurate. 2. YOLK MOTTLING AND OTHER EGG QUALITY CHARACTERISTICS* JACK L. FRY AND HENRY R. WILSON Poultry Science Department, Florida Agricultural Experiment Stations, Gainesville, Florida 32601 (Received for publication May 30, 1966)
Y
OLK mottling first received extensive study shortly after Nicarbazin was placed on the market; among the first studies reporting Nicarbazin mottling were those of Baker et al. (19S6) and Polin and Porter (1956). Several reports followed and Nicarbazin is now carefully kept out of layer feeds, but the problems it created have stimulated the interest in yolk defects. However, occasional severe cases of mot* Florida Agric. Exp. Sta. Journal Series, No. 2411.
\
tling have resulted in considerable economic loss to poultrymen and a corresponding inconvenience and loss to handlers and retailers. Beane et al. (1960) reported a brownish darkening of yolks due to the feeding of piperazine citrate. This report and field cases of yolk mottling in Florida and the Midwest prompted the initiation of the study herein reported. Recently, Beane et al. (1965) reported that three forms of piperazine (citrate, dihydrochloride, and sulfate) caused significantly greater yolk dis-
W. A. BECKER, J. V. SPENCER AND J. L. SWARTHOOD
wood, 1964. The pre-incubation storage of turkey eggs in closed environments. Poultry Sci. 43: 1526-1534. Cotterill, O. J., R. A. Gardner, E. M. Funk and F. E. Cunningham, 1958. Relationship between temperature and carbon dioxide loss from shell eggs. Poultry Sci. 37: 479-483. Funk, E. M., and J. Foward, 1960. Effect of holding temperature on hatchability of chicken eggs. Missouri Agr. Exp. Sta. Bull. 732. Krueger, W. F., W. E. McCune, C. B. Ryan, R. C. Jaska and J. H. Quisenberry, 1962. Optimum temperature and humidity for holding hatching eggs for short periods. Texas Agr. Exp. Sta. M. P. 592.
Landauer, W., 1961. The hatchability of chicken eggs as influenced by environment and heredity. University of Connecticut Monograph 1. McDonald, M. W., 1960. Effect of temperature of storage and age of fowl eggs on hatchability and sex ratio, growth, and viability of the chickens. Austral. J. Agr. Res. 11: 664-672. Proudfoot, F. G., 1964. The effects of plastic packaging and other treatments on hatching eggs. Canadian J. Anim. Sci. 44: 87-95. Proudfoot, F. G., 1965. The effect of film permeability and concentration of nitrogen, oxygen and helium gases on hatching eggs stored in polyethylene and Cryovac bags. Poultry Sci. 44: 636-644.
Response of the Reproductive Organs of the Japanese Quail to Pinealectomy and Melatonin Injections1 KAZUTAKA HOMMA2, LARRY Z. MCFARLAND AND WILBOR 0. WILSON Departments of Poultry Husbandry and. Anatomy, University of California, Davis, California 95616 (Received for publication May 12, 1966)
I
N recent years research on mammalian pineal physiology has been active. Melatonin elaborated by the pineal gland inhibits gonadal activity in rats (Wurtman et al, 1963a; Chu et al, 1964). The synthesis of melatonin in the rat pineal is controlled through environmental photoperioids, viz. it is accelerated by darkness and inhibited by light (Wurtman et al., 1963b). These diurnal changes in melatonin content of the pineal have been called the melatonin rhythm (Quay, 1964) Serotonin, the precursor of melatonin, also undergoes a marked diurnal change in the pineal (Quay, 1963), but there is no strict reciprocal correlation between the two rhythms. When rats were placed in continuous darkness or blinded, the melatonin 'Supported by research grant NB-04171 from National Institute of Neurological Diseases and Blindness. 2 Department of Animal Physiology, Faculty of Agriculture, Nagoya University, Anzyo, AichiKen, Japan.
rhythm was soon abolished, but the serotonin rhythm was maintained for more than 2 weeks, suggesting that the melatonin rhythm was under an exogenous control, whereas, serotonin rhythm was under an endogenous control (Snyder et al., 1964). Melatonin seems to play a role in controlling gonadal activity, since low doses of melatonin are more effective than serotonin in inhibiting ovarian activity of rats subjected to continuous light (Chu et al., 1964). Melatonin pharmacologically depresses the activity of the intestinal and uterine muscles with 300 [AM/ml. of melatonin completely inhibiting the spontaneous movements of rat uterine muscle in vitro (Hetrz-Eschel and Rahamimoff, 1965). Melatonin also acts as an antagonist toward certain types of chemical and auditory stimuli in the central nervous system ( Arutyunyan et al., 1964). Experiments on pineal physiology in avian species are not entirely in agreement with the results obtained in mammals. Con-
PINEALECTOMY AND MELATONIN INJECTIONS
trary to the results in rats, Axelrod et al., (1964) reported that the pineal enzyme HIOMT (hydroxyindole-O-methyl transferease), an enzyme necessary for melatonin synthesis, increased in activity as did the pineal weight in chickens placed in constant light. Chickens kept under diurnal photoperiods had higher HIOMT activity during the light period in contrast to that during the dark period. The high activities of this enzyme in an avian species (Quay, 1965) suggested the possibility that birds can produce larger amounts of melatonin than mammals. Furthermore, photoperiods favoring melatonin production are known to stimulate gonadal development in the chicken (Axelrod et al., 1964). The present experiments were designed to find out whether the pineal gland and melatonin are involved in the mechanism of photocontrol of the avian gonads. The Japanese quail (Coturnix coturnix japonica) was selected since gonad development of this species is known to be sensitive to environmental photoperiods (Wilson et al., 1962; Tanakae^aZ., 1965). MATERIALS AND METHODS
The source of Japanese quail and their general care were the same as reported previously (Tanaka et al., 1965). Pinealectomy. Two different procedures for pinealectomy were used depending upon the age of the birds. For quail younger than 3 weeks of age, high frequency electrocautery was used to destroy the pineal directly through the skull. This was feasible at this age, since ossification of the temporal bone was not advanced and the pineal body lay adjacent to the skull. After 4 weeks of age this procedure of pinealectomy was no longer applicable due to advanced ossification and the migration of the pineal deep between the cerebral hemispheres and cerebellum. In these birds the
315
bone was carefully removed using the dental drill and the pineal gland was surgically removed with its stalk. Local application of thrombin was used to minimize hemorrhage. The success of each pinealectomy was microscopically verified at autopsy. Only data from completely pinealectomized quail without brain damage were included in the analysis. The experiment involving length of photoperiod and pinealectomy comprised of a series of tests done at different seasons of the year and involving of separate environmental facilities. For this reason each group has its own control and the statistical analyses involved the t test rather than a more sophisticated statistical analysis. The effects of pinealectomy on gonadal growth were investigated in young quail of both sexes under 4 different photoperiods. Two were stimulatory photoperiods: continuous light (24 LL) and repeated cycles of 14 hours of light: 10 hours of dark (14L:10D). The other two light regimens were non-stimulatory (12L:12D, and 8L:16D), in that these photoperiods delayed gonadal growth for several weeks after hatch. Twenty to 36 quail were exposed to each photoperiod, approximately half of the quail from each photoperiod group were pinealectomized at 1 week of age, and the other half served as controls. All were killed at 4 weeks of age, and the gonads removed and weighed to the nearest 0.1 mg. In another experiment, quail were exposed to an 8L:16D photoperiod from hatch to 7 weeks of age. At 4 weeks of age, 13 males and 9 females were surgically pinealectomized, 17 males and 7 females were sham-operated, and 10 males and 7 females were used as non-operated controls. These birds were autopsied at 7 weeks of age. Melatonin treatments. In an experiment with young quail, the effect of exogenous melatonin was investigated by implanting
316
K. HOMMA, L. Z. MCFARLAND AND W. O. WILSON TABLE 1.—The results of four separate~tests designed to study the effects of pinealectomy at 1 week of age on body and gonadal weights of Japanese quail under four different photoperiods. Means + Standard error (number of samples) Photoperiods Body wt. at 4 wks. (g.)
Group
Testes wt. at 4 wks. (mg.)
Ovary wt. at 4 wks. (mg.)
Oviduct wt. at 4 wks. (mg.) 6 8 . 6 ± 1 0 . 2 (11) 6 3 . 2 + 1 1 . 0 (11)
Pinealectomy Control
24 L: O D 24 L: O D
92.6 + 1.4 92.5 + 2.2
654.3 + 65.2 (18) 599.5 + 74.5 (IS)
8 8 . 6 + 1 0 . 4 (19) 75.8+ 3.8 (17)
Pinealectomy Control
14 L: 10 D 14 L: 10 D
87.5 + 1.3*: 83.3 + 1.1
507.0 + 86.0 (11) 600.5 + 49.1 (13)
91.2 + 19.5 (9) 293.7±96.4 (9)* 65.6 + 5.9 (12) 121.9 + 38.9 (12)
Pinealectomy Control
12 L : 1 2 D 12 L:12 D
84.5 + 1.2 85.3 + 1.5
14.8 + 0.7 (8) 14.5 + 1.0 (12)
35.1 + 3.8 (6) 3 5 . 4 ± 3 . 6 (8)
17.3 + 1.4 (6) 15.5 + 0.8 (8)
Pinealectomy Control
8 L : 16 D 8L:16D
84.2 + 2.0*: 79.0 + 0.5
10.8 + 0.6 (12) 9 . 3 + 0 . 5 (13)
2 9 . 3 ± 3 . 2 (6) 22.6 + 2.1 (5)
17.4 + 0.5 (6) 16.7 + 1.5 (5)
: Means of body weights include both sexes. Means significantly (P<0.05) different from the control of the same photoperiod.
melatonin pellets. One hundred and twenty-six quail were divided into 4 groups soon after hatch. Each group was implanted at one week of age with small cylindrical 25 mg. pellets of cholesterol to which was added either 0, 1, 10, or 100 u,g. of melatonin. The average body weight of the quail at 1 week was approximately 18 g. The pellets were implanted subcutaneously on the dorsal aspect of the neck, after which time all birds were raised together under continuous light. They were killed at 4 weeks of age, at which time all the implanted birds still had a remnant of the pellet at the site of implantation. The effects of melatonin on the oviposition time were investigated in 10 laying quail. They were exposed to a 16L:8D photoperiod for 32 days. A record of egg production and time of lay was taken throughout this period. Every other day a TABLE 2.-
dye was administered orally (0.05 ml. of a Sudan III solution in corn oil) to each hen. Eggs were collected daily, boiled and sliced. Colored rings in the yolk were used as indices to estimate the number of maturing ovarian follicles. On day 21, 22, 23, and 24, these quail were injected subcutaneously with 1.0 mg. of melatonin dissolved in propylene glycol, and the record of egg production after melatonin treatment was compared with that preceding treatment. RESULTS AND DISCUSSION Pinealectomy. The gonadal and oviducal weights of 4 week old pinealectomized and control quail are shown in Table 1. Since the experiments involving various photoperiods shown in Table 1 are conducted in different seasons of the year and in different environmental facilities, considerable deviation can be expected from one
-Effects of pinealectomy at 4 weeks of age on body and gonadal weight of Japanese quail under 8 L: 16 D photoperiod. Means + Standard errors (number of samples) Body wt. (g)
Group Pinealectomy Sham operation Control
Male
Female
104.8 + 1.7(15) 104.8+1.4(13) 102.0 + 2.2(10)
104.5 + 1.6(9) 104.8 + 2.0(9) 99.0 + 2.5(7)
Testes wt. Ovary wt. Oviduct wt. at 7 wks. (mg.) at 7 wks. (mg.) at 7 wks. (mg.) 27.5 + 1.3(15) 38.9 + 4.6(9) 33.0 + 5.6(12) 41.6 + 3.0(9) 36.6+12.5(10) 45.7 + 6.1(7)
* Means are significantly (P<0.05) different from control.
20.1+2.2(9)* 19.2 + 1.5(9)* 30.4 + 5.2(7)
PINEALECTOMY AND MELATONIN INJECTIONS
317
TABLE 3.—Effects of 1,10 and 100 ng. melatonin implants made at 1 week of age on body and gonadal weight in young Japanese quail under continuous light. Means + Standard errors {number of samples) Body wt. at 4 wks. (g.) Group Male Control leg 10 ng 100 Mg
90.5 + 1.3(11) 87.1 + 5.1(22) 85.8 + 2.1(12) 83.4+1.2(18)
Female 87.1 + 82.7 + 89.3 + 86.4 +
Testes wt. at 4 wks. (mg.)
5.1(11) 5 8 8 . 6 1 2 9 . 7 " ^ , 2.2(15) 279.9 + 4 9 . 8 * ^ a 2.9(21) 400.5 + 80.4 ~ - > a 2.0(16) 598.4 + 7 4 . 5 ^
Ovary wt. at 4 wks. (rag.)
Oviduct wt. at 4 wks. (mg.)
89.1+6.7 65.5 + 5.8* 76.5±6.8 81.2 + 7.8
82.7 + 12.3 46.1 + 6.0* 83.8+17.5 68.3 + 11.7
' Means are significantly (P<0.05) different from control. Means are significantly different ( P < 0 . 0 5 ) .
test to another; thus, it was thought desirable to analyze the data by a separate t test for each photoperiod rather than by analysis of variance of the whole data. Under the three types of photoperiods, i.e., 24 LL, 12L:12D, and 8L:16D, pinealectomized quail showed no statistically significant difference either in gonadal or oviducal weight from that of the controls. Under 14L:10D photoperiod, pinealectomized females showed a statistically significant increase in oviducal weight ( p < 0.05), but the slight increase in ovarian weight in the pinealectomized birds was not significant. The gonadal development of quail operated on at 4 weeks and killed at 7 weeks of age is shown in Table 2. In this experiment, both pinealectomy and sham operation caused a comparative decrease in gonadal weight (not significantly different from control), and in oviducal weight (significantly different from control, p < 0.05). Although there was no sign of retardation of body growth in the two operated groups, the results suggest that retardation of oviducal growth in the pinealectomized birds was induced by surgical stress and not by removal of the pineal gland.
tardation of gonadal and oviducal growth (p < 0.05). However, with increasing amounts of melatonin the gonadal inhibition was decreased rather than increased. At the highest dose of 100 \i.g., melatonin apparently had no gonadal inhibitory effect. This type of dose-response relationship can be expected only when gonadal inhibitory effect of melatonin is indirect and could offer an explanation for the high HIOMT activity of the pineal gland in sexually active birds of various species (Quay, 1965). The results of melatonin injection into laying quail are shown in Figure 1. The time of injection is indicated by arrow (1700 hours). The distribution of the time of lay was based on number of eggs collected during a 22 day period prior to the melatonin injection (170 eggs), and during a 10 day period following melatonin injection (95 eggs). Melatonin injection significantly (p <0.01) affected the time of lay (Chi-square test, p < 0 . 0 1 ) . The major peak of lay at 2100 hours prior to the treatment was advanced 2 hours following melatonin. Furthermore, some new minor peaks appeared after melatonin treatment. A daily dose of 1 mg. did not affect the number of developing follicles in the ovary (Table 4), and no changes in the Melatonin implantation. The effects of behavior of the birds were observed. exogenous melatonin on gonadal weight in The results of the present experiments young quail under continuous light are favor the view that the pineal is involved in shown in Table 3. Quail implanted with the mechanisms regulating gonadal func1 jtg. of melatonin showed a significant re- tion in the quail, presumably through se-
31S
K. HOMMA, L. Z. MCFARLAND AND W. O. WILSON
^o PRIOR TO TREATMENT FOLLOWING TREATMENT
<
S20 < O
0
2
4
6
8
10 12 14 16 TIME IN HOURS
18 2 0 22 24
LIGHT OFF
LIGHT ON
FIG. 1. Distribution of eggs laid by quail before and after melatonin injections. Time is expressed on 24 hour clock.
cretion of melatonin. Undoubtedly, the pineal is related to certain pathways which transmit environmental stimuli to the pituitary, but the participation of the pineal is not strong enough to modify the entire gonadal response to various environmental stimuli, i.e., pinealectomy did not restore the gonadal growth of those quail raised under non-stimulatory photoperiods. One of the important roles of the pineal would be that of a monitor which prevents hypersecretion of pituitary hormones (Moszkowska, 1965), or that of "Zeitgeber" or
timing device which regulates the time of hormone release as was indicated in the experiments of laying quail (Figure 1). Shellabarger reported that pinealectomy in the chicken caused gonadal hypertrophy in old cockerels (1952) and gonadal inhibition in young cockerels (1953). It appears from his results that the function of the pineal is influenced by age as well as by environmental factors. Such an age effect was not evident in quail under a non-stimulatory photoperiod (8L:16D). The mechanism whereby melatonin causes gonadal inhibition in mammals is still not fully understood. A direct inhibition of ovarian activity has been proposed, based on the evidence that labeled melatonin was accumulated by the rat ovary (Wurtman et al., 1964). This would not appear to be the case in quail, since higher doses of melatonin were not more inhibitory (Table 3). SUMMARY Pinealectomy of young coturnix quail permitted rapid oviducal growth in the female under 14L:10D photoperiod, but was without effect in quail kept under nonstimulatory photoperiods. Melatonin implantation in 1 week old birds at a level of 1 u.g. per bird inhibited gonadal growth, while higher levels were less effective. Subcutaneous injection of 1 mg. of melatonin for 4 days changes the distribution of the time of lay, but did not inhibit ovarian activity. REFERENCES
TABLE 4.—Number of developing follicles in the ovary before and after melatonin injection 1 mg.j'day/bird for 4 days)
Before injection After injection
N
Number of observation
«™£* of nj^Ajev (Mean+S.E.).
36 30
3.4 + 0.3 3.3 + 0.3
Arutyunyan, G. S., M. D. Mashkovskii and L. F. Roshchina, 1964. Pharmacology of melatonin. Fed. Proc. 23 :T. 1330-1332. Axelrod, J., R. J. Wurtman and C. W. Winget, 1964. Melatonin synthesis in the hen pineal gland and its control by light. Nature, 201: 1134. Chu, E. W., R. J. Wurtman and J. Axelrod, 1964. An inhibitory effect of melatonin on the es-
PlNEALECTOMY AND MELATONIN INJECTIONS trous phase of the estrus cycle in rodents. Endocrinology, 75: 238-242. Hertz-Eschel, M., and R. Rahamimoff, 1965. Effects of melatonin on uterine contractivity. LifeSci. 4 : 1367-1372. Moszkowska, A., 1965. Contribution a l'etude du mecanisme de 1'antagonisme epiphyso-hypophysaire. In Structure and Function of the Epiphysis Cerebri. Ed. Aliens Kappers, J., and J. P. Schade. pp. 564-576, Elsevier Pub. Co., Amsterdam. Quay, W. A., 1963. Circadian rhythm in rat pineal serotonin and its modification by estrous cycle and photoperiod. Gen. Comp. Endocr. 3 : 473-479. Quay, W. A., 1964. Circadian and estrous rhythms in pineal melatonin and 5-hydroxyindole-3-acetic acid. Proc. Soc. Exp. Biol. Med. 115: 710-713. Quay, W. A., 1965. Retinal and pineal hydroxyindole-O-methyl transferase. Life Sci. 4 : 983991. Shellabarger, C. J., 1952. Pinealectomy vs. pineal injection in the young cockerel. Endocrinology, 5 1 : 151-154. Shellabarger, C. J., 1953. Observation of the pi-
319
neal in the White Leghorn capon and cockerel. Poultry Sci. 32 : 189-197. Snyder, S. H., M. Zweig and J. Axelrod, 1964. Control of the circadian rhythm in serotonin content of the rat pineal gland. Life Sci. 3 : 1175-1179. Tanaka, K., F. B. Mather, W. 0. Wilson and L. Z. McFarland, 1965. Effect of photoperiods on early growth of gonads and on potency of gonadotropins of the anterior pituitary in coturnix. Poultry Sci. 44: 662-665. Wilson, W. O., H. Abplanalp and L. Arrington, 1962. Sexual development of coturnix as abfected by changes in photoperiod. British Poultry Sci. 3 : 105-113. Wurtman, R. J., J. Axelrod and E. Chu, 1963a. Melatonin, a pineal substance: Effect on the rat ovary. Science, 141: 277-278. Wurtman, R. J., J. Axelrod and L. S. Phillips, 1963b. Melatonin synthesis in the pineal gland controlled by light. Science, 142: 1071-1073. Wurtman, R. J., J. Axelrod and L. R. Potter, 1964. The uptake of H3-melatonin in endocrine and nervous tissues and the effects of constant light exposure. J. Pharmacol. Exp. Ther. 143:314-318.
Studies of Dietary Piperazine, Phenothiazme, and Dibutyltin Dilaurate. 2. YOLK MOTTLING AND OTHER EGG QUALITY CHARACTERISTICS* JACK L. FRY AND HENRY R. WILSON Poultry Science Department, Florida Agricultural Experiment Stations, Gainesville, Florida 32601 (Received for publication May 30, 1966)
Y
OLK mottling first received extensive study shortly after Nicarbazin was placed on the market; among the first studies reporting Nicarbazin mottling were those of Baker et al. (19S6) and Polin and Porter (1956). Several reports followed and Nicarbazin is now carefully kept out of layer feeds, but the problems it created have stimulated the interest in yolk defects. However, occasional severe cases of mot* Florida Agric. Exp. Sta. Journal Series, No. 2411.
\
tling have resulted in considerable economic loss to poultrymen and a corresponding inconvenience and loss to handlers and retailers. Beane et al. (1960) reported a brownish darkening of yolks due to the feeding of piperazine citrate. This report and field cases of yolk mottling in Florida and the Midwest prompted the initiation of the study herein reported. Recently, Beane et al. (1965) reported that three forms of piperazine (citrate, dihydrochloride, and sulfate) caused significantly greater yolk dis-