Effects of exogenous melatonin on some endocrine, behavioural and metabolic parameters in japanese quail Coturnix coturnix japonica

(bmp. Biochem.Physid

0300-9629/93 66.00+0.00 ‘c 1993 P~,-~~Ot, Press Ltd

Vol. IOSA,No. 2,pp. 323-328.1993

Printedin Great Britain

EFFECTS OF EXOGENOUS MELATONIN ON SOME ENDOCRINE, BEHAVIOURAL AND METABOLIC PARAMETERS IN JAPANESE QUAIL COTURNIX COTURNIX JAPONICA M. ZEMAN,*t P.

WBOH,

M. JURANI,D. LAMOSOVA, L. KoSTAL, B. BILCIK,P. BLA~ICEK~and E. JURANIOVA tlnstitute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, lvanka pri Dunaji. Slovakia; IMilitary Hospital, Bratislava, Slovakia (Received 28 July 1992; accepted

I 1 September 1992)

Abstract-l, Melatonin administration in drinking water (5 pg/ml) to Japanese quail resulted in a 20-fold increase of plasma melatonin levels in comparison with the control, day time concentration (0.34 + 0.05 vs 6.88 + 1.10nmol/l). 2. Plasma triiodothyronine levels increased (5.8 + 0.93 vs 7.97 + 0.64 nmol/l), corticosterone decreased (28.04 f 3.42 vs 15.96 _+2.56 nmol/l) and no significant changes were recorded in thyroxine concentration after the treatment. 3. A higher occurrence of sleeping and lower occurrence of pecking were found in melatonin treated quail. 4. Abdominal fat deposition as well as the content of total lipids in the breast muscle and triacylglycerols in plasma were decreased in treated birds indicating an inhibitory effect of melatonin on lipogenesis. 5. Melatonin increased RNA content in the breast muscle but did not affect plasma glucose concentration and body weight.

INTRODUCTION Tile pineal hormone melatonin is implicated in the control of seasonal cycles and transmits information or the photoperiod to the neuroendocrine-gonadal axis in photoperiodic mammals. In birds, a role of the pineal gland in the control of reproduction is still obscure (Gwinner, 1981, 1989). The pineal gland sec’ms to be involved in the control of avian circadian rhlthms, but the effects differ considerably among different taxonomic groups. Pinealectomy disrupts tht. circadian rhythms of the locomotor activity in the house sparrow (Gaston and Menaker, 1968), leads to rh:%hm disturbances in the starling (Gwinner et al., 19X7) but has no detectable effects on the rhythms in gallinaceous birds (Simpson and Follett, 1981). n addition to a role in the circadian organization, melatonin and the pineal gland can influence a wide rar ge of physiological and behavioural processes. An int -aperitoneal injection of melatonin induces a sleep state in young chicken (Hishikawa et al., 1969). Fu -thermore, pinealectomy impairs body weight gain and efficiency of energy retention while exogenous meiatonin increases body gain, gain:feed ratio and energy retention in broiler chicks (Osei et al., 1989). Melatonin may exert its effects either directly or through interactions with other hormones involved in growth control, particularly thyroid and steroid hor*To whom all correspondence CBPA

IW--1

should be addressed.

mones (Oishi and Lauber, 1974; Cogburn and Harrison, 1980; Binkley, 1988). However, results are often contradictory and the mechanisms remain poorly understood. The aim of the present studies was to investigate the influence of exogenous melatonin on the physiological processes connected with the growth in Japanese quail (Coturnix coturnix japonica) from hatching until 24 days of age (a time selected to avoid intersexual differences in body weight). Changes in selected behavioural activities, circulating concentrations of melatonin, thyroid hormones and corticosterone as well as certain parameters of lipid, protein and carbohydrate metabolism following melatonin treatment were studied. MATERIALSAND

METHODS

Japanese quail hatchlings of both sexes were weighed and assigned to two groups of 10 birds. They were individually wing-banded and placed in boxes in a brooding room. The ambient temperature in the room was kept at 37°C for the first 2 days and then decreased stepwise by 3°C at 4 day intervals to reach 24°C at the end of week 3. Continuous lighting was provided by cool white fluorescent tubes that produced a light intensity of 40-60 lx at the level of the birds heads. The starter feeding mash for young turkeys (27% crude protein and 13.7 kJ of metaboliz able energy/kg) and water were provided ad fib. 323

M.

324

ZEMANet al.

0

pooled

0

y = -0 94x + 1.68 standard y = -0.95x + 1.42

blood

plasma

i -0.6

-0.4

-0.2

0.0

0.2

04

0.6

1.0

0.8 1ogit

Fig. I. Parallelism between serially diluted melatonin standard and pooled blood plasma collected in the middle

of the dark

period

(LD 12: 12).

Melatonin (Sigma, St Louis, MO, U.S.A.) was administered in drinking water at a concentration of 5 pg/ml. The stock melatonin solution was prepared by dissolving 10 mg of melatonin in 1 ml of 95% ethanol and was kept at 4°C. The fresh drinking water was prepared and changed daily by dilution of the stock solution with tap water. The control birds received the same amount of ethanol in drinking water as the experimental birds. During the whole experiment individual body weight and food consumption per group were recorded. Body weight gain and feed : gain ratio were determined at 3-4 day intervals. At 21 days of age the behaviour of both groups of quail was observed for the whole 24 hr cycle. The behavioural observations were made during the first 20 min of each hour. Each bird was marked by a number on its back and the behaviour of each quail was recorded every 2 min using “on-the-dot” sampling methods (Slater, 1978). Eight behavioural categories were recorded: drinking, feeding, pecking, preening, sitting, sleeping, standing and walking. On the basis of the behavioural records the percentage time spent performing each single activity during each hour and during the whole day were calculated. At 24 days of age the quail were decapitated and blood was collected into heparinized tubes. The blood samples were centrifuged for 10 min at 3000 g at 4°C and plasma was stored at -20°C until analyses were

performed. The weight of liver and abdominal fat were recorded. The content of total proteins (Lowry et al., 1951) total lipids, glucose (kits Lachema Bmo, CSFR), triacylglycerols (kit, Boehringer Mannheim, Dormstadt, FRG) and polyunsaturated fatty acids (Barash and Akow, 1987) were measured in plasma. A sample of breast muscle was taken for the determination of total lipids, total proteins, RNA and DNA (Pechan, 1966). The dry weight of breast muscle was determined by drying at 105°C until constant weight. Thyroxine (T4) and triiodothyronine (T3) were measured by direct RIA without extraction (Fiildes et al., 1978). Corticosterone was assayed after extraction with methylene chloride (Satterlee and Johnson, 1988) and melatonin was estimated in plasma by direct radioimmunoassay (Frazer et al., 1983). Melatonin (3H) (3.15 TBq/mmol) was purchased from the Radiochemical Centre Amersham, U.K. and the melatonin antiserum was kindly provided by Stockgrand Ltd., Department of Biochemistry, University of Surrey, U.K. The assay was validated for quail plasma by parallelism. No statistically significant difference was noted between the slopes of the serially diluted melatonin standard and a pooled blood plasma collected in the middle of the dark period from quail kept on LD 12: 12 (Fig. 1). The statistical significance of differences induced by the melatonin treatment was evaluated by Student’s t-test.

Table I. Effect of melatonin in drinking water on plasma melatonin, T3, T4 and corticosterone levels (nmol/l) in 24-day-old Japanese quail

RESULTS

Control Melatonin Triiodothyronine Thyroxine Corticosterone

0.34 + 5.86+ 8.08 + 28.04 +

0.05 I.12 0.93 3.42

Each value represents the mean of IO fP

< 0.001.

Melatonin 6.88 * I.107 1.97 f 0.64. 6.44 +_I .26 15.96 * 2.56’

quailf

SEM.

‘P ~0.05;

The administration of melatonin in drinking water was reflected in concentrations of plasma hormones. In particular these changes were apparent in plasma melatonin, with levels being about 20-fold higher in the experimental group in comparison with the controls (Table 1). Plasma T3 levels increased after the melatonin treatment (P < 0.05) and no significant

Melatonin

effects in quail

325

40 ,

2 * -d

!

0 control KQI melatonin

Fig. 2. Effect of melatonin in drinking water on mean proportion (%) of time spent per bird in different behavioural activities in 24 hr of observation. Each value represents the mean of 10 quail & SEM. “P < 0.05; cP < 0.001.

changes were recorded in T4 concentrations. Plasma corticosterone levels showed a significant (P < 0.05) decrease in the treated birds. No apparent nycthemeral changes were found in any observed behavioural patterns. Sleeping and feeding took the greatest portion of the time in both groups, as can be seen from the cumulative data from al 24 behavioural samples taken through the day (Fig. 2). A higher proportion of the time spent sleeping was found in melatonin treated quail in comparison with controls (P < 0.001). By contrast, the occurrence of pecking was significantly lower in the treated group (P < 0.01). Melatonin had no effect or the time spent walking, standing, sitting, preening and drinking. Melatonin administration did not influence body wtight gain, feed consumption and gain: feed ratio (Table 2). However, there was a significant (P < 0.05) increase in muscle RNA content in breast muscle and an increase in DNA was significant at the level P = 0.08 (Table 3). Despite these indications of an effect of melatonin on protein metabolism, the protein content in the breast muscle, and the protei? : DNA and RNA : DNA ratios were unaffected by melatonin treatment. Melatonin influenced lipid metabolism. We failed Table 2. Effect of melatonin in drinking water on the performance of Japanese quail during 24 day period Bodv wei& (a) Weight & (3 Feed intake (g) Feed:gain ratio

Control

Melatonin

84.62 + 3.38 78.59 ? 3.38 242.90 3.06

91.57 + 3.19 85.42 ; 3.16 249.90 2.93

Each value represents the mean of 10 quail + SEM.

to identify any abdominal

fat in melatonin treated birds while the fat content was 30.9 f 9.0 mg in control quail. The significant decrease in total lipids in breast muscle and triacylglycerols in plasma were found after treatment while the decrease in liver weight was not significant (Table 4). Melatonin did not affect concentrations of polyunsaturated fatty acids, total lipids, cholesterol, and glucose in plasma.

The amount of melatonin given in the drinking water in our experiment was relatively low in comparison with the doses administered directly into the mouth-5 mg/bird (Dittami and Janik, 1986), in drinking water-l mg/ml (Binkley and Mosher, 1985), in the feed-15 mg/kg (Osei et al., 1989) or by implants of Silastic tubes (Turek and Wolfson, 1978; Beldhuis et al., 1988) or beeswax pellets (John et al., 1986, 1990). Plasma melatonin concentrations have only been measured after Silastic tubes implantation and were found to be about two orders higher than high dark time values (Beldhuis et al., 1988). Plasma Table 3. Effect of melatonin in drinking waler on musculus pectoralis composition of 24-day-old Japanese quail

Control Dry weight (%) Total protein (mg/g wet tissue) DNA (mgh) RNA (mg/g) Protein/DNA RNA/DNA Total lipids (mg/g wet tissue)

45.46 f 1.41 159.95 f 5.06 2.8 I * 0.09 19.54 + 0.54 57.51 f 2.82 7.02 f 0.21 6.20 & 0.40

Mclatonin 46.31 f 1.45 171.40 * 7.10 3.12kO.14 21.76 f 0.71’ 55.71 + 2.84 7.04 f 0.19 5.09 f 0.34’

Each value represents the mean of IO quail f SEM. lP -z 0.05.

M. ZEMAN et al.

326

Table 4. Effect of melatonin in drinking water on liver weight, and the lipid metabolism parameters in 24-day-old Japanese quail Control Liver (g) Total lipids in plasma (g/l) Cholesterol in plasma (mmol/l) Triacylglycerols in plasma (mmoli) Polyunsaturated fatty acids in plasma (mmol:l) Glucose (mmol/l) Each value represents

3.29 3.28 3.04 3.01

k + * f

0.17 0.53 0.20 0.20

Melatonin 2.80 2.83 3.22 2.56

f + k *

0.14 0.42 0.16 0.19’

1.35+_0.16

1.26iO.10

17.47 + 0.21

17.70 i 0.28

the mean of IO quail + SEM. *P < 0.01.

melatonin concentrations in our experiment were about 4-fold higher than usually found during the middle of the dark period. The pooled blood plasma sample of control quail collected in the middle of the dark period and measured in the same assay as samples from the experiment contained 1.81 k 0.21 nmol of melatonin per ml. This concentration corresponds with findings of other authors (Underwood et al., 1984; Meyer and Millam, 1991) who found high nightime melatonin concentrations in the range 400-600 pg/ml (1.722-2.583 nmoljl). Quail were kept in continuous light throughout the whole experiment. It is well known that light suppresses melatonin synthesis in mammals (Illnerova et al., 1978) and birds (Binkley et al., 1977; Vakkuri et al., 1985). Melatonin concentrations in control quail were low indicating that the light intensity used in our experiment was sufficient to suppress melatonin synthesis, and that endogenous melatonin did not interfere with the treatment. Growth promoting effects of melatonin have been suggested by several authors (McKeown ef al., 1975; Darre et al., 1980; Osei et al., 1989; John et al., 1990). However, the possible mechanism of this action on growth regulation is not understood. Melatonin may influence body weight gain by improving the efficiency of energy retention in chicken (Osei et al., 1989). These authors showed that the growth suppressing effect of pinealectomy was due to a decrease in efficiency of energy retention, since feed consumption remained unaffected. Chicken kept in continuous light exhibit similar negative effects on energy retention, as pinealectomized birds. While pinealectomy significantly depressed growth (0~01 et al., 1980; Osei et al., 1989) it was not found to affect the basal metabolic rate. Melatonin may improve the energy balance at several levels. In our experiment a higher occurrence of sleeping was observed in treated birds in comparison with controls during a 24 hour period. The treated birds thus expended less energy on physical activity than control ones. Pecking is generally considered as one kind of stereotypic behaviour. The decreased frequency of this activity in treated birds suggests a sedative action of administered melatonin. The sedative effect of melatonin was reported after using high pharmacological doses in birds (Hishikava et al., 1969; Hendel and Turek, 1978; Binkley, 1988) while a substantially lower dose was used in our experiment.

Another level at which melatonin may exert its effects on growth is through an alteration of intermediary metabolism which is controlled by hormones. Several studies deal with the effects of pinealectomy or melatonin administration on thyroid gland function. In mammals, exogenous melatonin exerts mainly negative effects on thyroid function (Vaughan et al., 1984). In birds, the role of the pineal gland in this respect is not clear since opposite results are frequently found. A significant decrease of thyroid weight in pinealectomized S-week-old quail (Oishi and Lauber, 1974) but not in chicken (0~01 er al., 1980) have been found. One source of discrepancy in the studies on the influence of the pineal on plasma thyroid hormones has been discussed by Sharp et al. (1984). In their extensive study an inhibitory effect of pinealectomy on thyroid hormones levels was found only under constant lighting conditions. They concluded that in a light-dark cycle the amplitude of changes in thyroid hormone concentrations induced by feeding pattern and imposed by the lightdark cycle is so pronounced that any effects of the pineal are masked. Our results of increased T3 levels in young quail kept in constant light and given by exogenous melatonin support this conclusion. Dietary melatonin increases plasma concentrations of T3 and T4 in chicken (Osei et al., 1989). Plasma T3 concentrations appear to be positively correlated with growth rate in chicken (Kuhn et al., 1982) and a higher concentration of this hormone may be partly responsible for the increase in efficiency of protein deposition in younger chicks (Decuypere and Buyse, 1988). Exogenous corticosterone is a potent lipogenic agent in chicken (Decuypere and Buyse, 1988, for review) and it can depress circulating T3 levels (Buyse et al., 1987). The decreased level of this dominant avian glucocorticoid after the melatonin treatment accords with the decreased fat deposition and the stimulated protein metabolism in treated quail. The recorded changes in plasma hormones, especially the increase in T3 and the decrease in corticosterone levels, indicate the involvement of the endocrine system in mediating melatonin effects on the intermediary metabolism in birds. Polyunsaturated fatty acids were not changed after treatment indicating that lipolysis was not affected by the melatonin. The decreased content of triacylglycerols in plasma together with the decreased deposition of the abdominal fat and the decreased lipid content in the breast muscle suggest inhibited lipogenesis, especially in female quail. The increased content of RNA and the trend towards the increased content of DNA in breast muscle indicate proteoanabolic effects of melatonin treatment and together with the decreased lipid content suggests some “repartitioning” effects of melatonin in quail. The present results show that melatonin can influence the quail performance at several levels. Exogenous melatonin modulated behaviour. plasma

Melatonin concentrations

of several

peripheral

hormones

effects in quail

and

In comparison with the central role of melatonin in the control of reproduction in photoperiodic mammals, there may be more general effects in birds. intermediary

metabolism

A :knowledgemenf-The Arendt (Stockgrand L niversity of Surrey, SCrum.

of

lipids

and

protein.

authors are grateful to Dr J. Ltd., Department of Biochemistry, U.K.) for the gift of melatonin anti-

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