INTRODUCTION

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THE PINEAL ORGAN AND ITS metabolites play an important role in circadian thermoregulatory adjustments of body temperature (T_b) in many species. Pinealectomy resulted in the loss of endogenous circadian rhythm of locomotor activity in the house sparrow and abolished T_b rhythm in sparrows kept in complete darkness (4, 12). Melatonin (Mel) administration lowers T_b by 2-3°C in mice (9). This latter observation correlates with the reduction in plasma L-thyroxine concentration observed in pinealectomized White Leghorn roosters, suggesting a possible link between Mel and regulation of metabolic rate (16). Pinealectomy or blinding did not abolish the circadian rhythm of locomotor activity in pigeons; however, when these treatments were combined, this rhythm disappeared under constant or dim light (10). In pigeons, Mel is synthesized not only by the pineal gland but also in the retina (21, 27). Accordingly, daily administration of Mel to pinealectomized and blinded pigeons restored circadian rhythms of locomotor activity, and T_b was correlated to Mel injections (20).

In Japanese quails, exposure to cold lowered plasma, pineal, and retinal Mel concentrations (17); in contrast, cold stress increased Mel levels in the pineal gland of rodents (18, 26). The circadian rhythm of T_b has been suggested to be related mainly to heat-loss mechanisms (24), because in mammals T_b preserves its circadian oscillation, even when heat production does not oscillate (11, 23). To the best of our knowledge, there are no data related to thermoregulatory responses leading to hypothermia in response to Mel administration. Nor is there any information regarding the involvement of Mel in thermoregulatory responses under conditions of heat stress. In the present study, we examined the effect of Mel on thermoregulatory mechanisms and cardiorespiratory responses in birds maintained at thermoneutrality or heat- or cold-stress states.

	MATERIALS AND METHODS

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Experimental Procedure

Experimental Protocols

Experiment 2. Hens (n = 36) were exposed to either acute heat (37°C) or cold (10°C) stress for 5 h. In each thermal treatment, NMP-vehicle controls were compared with hens injected with 160 mg Mel/kg body wt; injection was performed either immediately before or 30 min after the start of heat or cold exposure.

Experiment 3. Twelve hens divided equally into two groups, NMP controls and hens injected with 160 mg Mel/kg body wt, were held under thermoneutral conditions. Body, metatarsal, comb, and feathered trunk-skin temperatures; respiratory rate; oxygen consumption; evaporative water loss; heart rate; and blood pressure were recorded 30 min before injection and every 10 min after injection until termination of the experiment.

Data Analysis

	RESULTS

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Experiment 1: Thermoneutral State

View larger version (28K): [in this window] [in a new window]   Fig. 1.   A: dose response to melatonin (Mel) dissolved in ethanol [0 (), 5 (), 10 (), 20 (), 40 (), 80 (+), and 160 mg Mel/kg body wt ()] on body temperature of White Leghorn hens. B: dose response to Mel dissolved in N-methyl-2-pyrrolidone (NMP) [untreated control (), NMP-treated control (), 80 (+), and 160 mg Mel/kg body wt ()]. Points represent means ± SE.

Experiment 2: Cold and Heat Stresses

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effect of Mel administration before and after ambient temperature exposure. A: 10°C. B: 37°C. [NMP-treated control (), 160 mg Mel/kg body wt injected 5 min before () or 30 min after () temperature exposure.] Points represent means ± SE.

Experiment 3: Thermal and Cardiorespiratory Responses

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of Mel administration (160 mg/kg body wt) on body temperature (A), feathered-skin temperature (B), and unfeathered-organ temperatures [comb (C), shank (D)]. Points represent means ± SE.

Mel induced an immediate rise in respiratory rate (Fig. 4A, P < 0.05), which peaked 30 min after injection, then dropped, reaching a basal level at 100 min, and stabilized. The pattern of evaporative heat loss was similar to that of respiratory rate. It rose (P < 0.05) immediately after Mel injection, decreased, then climbed back up to initial values 60 min later (Fig. 4B). During the first 60 min, metabolic heat production in Mel-treated hens increased by ~7% above the control level, but this rise was not statistically significant. Heat production then decreased and stabilized toward the end of the experiment (Fig. 4C). In control hens, heat production was stable during the first 60 min, then rose slightly. The different heat production patterns in control vs. Mel hens (ascending and descending patterns, respectively) was reflected in a treatment-by-time interaction (P < 0.05). Mel induced a transient rise in heart rate immediately after injection, which was followed by a decrease (P < 0.05) of ~10% thereafter (Fig. 5A). Mean blood pressure (Fig. 5B) was not altered during the first 60 min; it then rose (P < 0.05) in Mel-treated birds by ~10% above control values.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of Mel administration (160 mg/kg body wt) on respiratory rate (A), evaporative heat loss (B), and heat production (C). Points represent means ± SE. Bw, body wt.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Effect of Mel administration (160 mg/kg body wt) on heart rate (A) and mean blood pressure (BP; B). Points represent means ± SE.

	DISCUSSION

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A single injection of Mel caused a significant dose-dependent hypothermic response in laying hens. Such a response to Mel has been found previously in sparrows (5, 12), chicks (2), and several mammalian species (15, 22), including humans (6, 7, 8, 25). The present study revealed two phases following Mel injection: the first lasted 1 h, during which the maximal hypothermic response occurred associated with enhancement of heat-loss mechanisms; the second phase lasted at least 3 h, during which the birds were hypothermic but heat-loss mechanisms were no longer active. The hypothermic response was not related to the type of Mel-dissolving vehicle, because neither vehicle induced hypothermia on its own. The use of NMP as a solvent in vivo has been previously reported (3).

This study showed that heat-loss mechanisms are responsible for Mel-induced hypothermia. Peripheral vasodilatation (indicated by the 3-5°C rise in skin temperature and recorded only in the unfeathered-skin extremities) caused a marked rise in nonevaporative heat loss. In contrast, under thermoneutral conditions Mel did not increase feathered-skin temperatures of the body trunk above initial values (38°C), the latter being higher than those in the comb and metatarsus. In fact, convective and conductive heat loss from the skin seemed to be the major heat-loss pathway inducing hypothermia. Although respiration rate rose to 140 breaths/min, evaporative water loss rose in Mel hens by only ~13% above control values during the first hour after Mel injection; this value was much lower than expected for panting White Leghorn hens (1, 28). Moreover, the extra heat loss by evaporation during the first hour after Mel injection (2.9 W/kg body wt) was similar to the slight rise (3.0 W/kg body wt; NS) in heat production recorded during the same time period; the latter was due to the increased respiratory-muscle activity during panting. It is not clear, however, why enhanced respiratory evaporation induced by Mel is ineffective in terms of heat loss.

In agreement with the finding that nonevaporative heat loss is the main heat-loss pathway causing hypothermia in Mel birds, we found that under cold conditions (Fig. 2A) T_b reached a nadir of ~1°C lower than under thermoneutral conditions (Fig. 1, A and B). This can only be attributed to an increase in sensible heat loss from the skin under cold conditions; as indicated, panting was not observed in cold-stressed Mel hens. In Japanese quails, exposure to cold significantly lowered plasma, pineal, and retinal Mel levels and depressed the usual Mel rise in the dark (17). The latter parameters had been suggested to be a manifestation of a natural mechanism that protects the animal from severe hypothermia during cold nights.

Of particular interest are the responses to Mel under heat stress. Mel administered 30 min after the start of heat exposure did not cause the typical immediate hypothermia (Fig. 2B). This is probably because heat exposure induced skin vasodilatation and panting that Mel injected 30 min later could not enhance further. In contrast, in birds injected with Mel just before the start of heat exposure, the immediate skin vasodilatation and rise in respiratory rate induced hypothermia even though hens were exposed to heat. It is worth noting that under heat stress, Mel lowered T_b below control values not only during the first hour after Mel injection but also during the last 2 h of the experiment. This finding could be developed into a tool to prevent or reduce the heavy losses of domestic fowl during hot spells in hot countries, provided that Mel is administered before air temperatures rise.

The pattern of heat production in response to Mel followed that of T_b, except during the first hour after Mel injection. During that time heat production did not fall with T_b because of panting activity. However, later, when panting ceased, the drop in heat production followed the reduction in T_b (Q₁₀ effect). The rise in heat production after 140 min followed a similar rise in T_b. An almost similar pattern was noted for heart rate as well (Fig. 5A). The similar responses were expected and it is well established that a rise in metabolic rate is associated with a rise in heart rate, representing a rise in cardiac output. The 15% drop in heart rate occurred when metabolic rate was at a minimum, and later (after 140 min), the rise in heart rate corresponded to the increase in both T_b and heat production. The slight rise in arterial blood pressure in Mel hens relative to controls probably represented a reflex response to the decline in heart rate (Fig. 5B).

This study shows that a single injection of a pharmacological dose of Mel affects the control mechanism of thermoregulation. Our results, combined with previous observations on circadian changes in T_b associated with plasma Mel level, suggest a mechanism for the nocturnal decline in T_b. The central control of thermoregulation involves several peptides and monoamines that act in the preoptic and septal areas in birds and mammals (13, 14). Mel might affect thermoregulation by acting in the brain (24); however, peripheral action of Mel should not be ruled out. Further studies are needed to explore this hypothesis. Furthermore, in the present study Mel induced hypothermia during the morning and early afternoon hours, when endogenous Mel secretion is low; responses may change if Mel is administered at night hours when Mel secretion is high.

In relation to a "set-point" model, Mel may act by lowering the central set-point temperature, thereby causing immediate stimulation of heat-loss mechanisms. These immediate changes, which last about an hour, are long enough to induce deep hypothermia, which then lasts for several additional hours.