Indian Journal of Experimental Biology Vol. 38, November 2000, pp. 1111-1116

Influence of photoperiods on glycemic and adrenal catecholmninergic responses to melatonin administrations in adult male roseringed parakeets,

Psittacula krmneri Neumann

S K Maitra, M Dey, R Dey, S Bhattacharya & A Sengupta

Department of Zoology, University of Burdwan, Golapbag, Burdwan 713 I 04, Indi a

Received 9 August /999: revised 21 June 2000

Effects o f daily (one hour prior to onset of darkness) injection of melatonin (25 J..lgl I 00 g body wt. for 30 days) on concen trations of blood glucose and adrenal catecholamines were studied in adult male roseringed parakeets, P. krameri under both natural (NP; about 12L: 120) and artific ial long (LP; 16L: 80; lights were available in between 0600 and 2200 hrs) or short (SP; 8L: 160; lights were available between 0600 and 1400 hrs) photoperiodic conditions . The results indicate that neither LP, nor SP as such exerts any significant effect on blood glucose titre of contro l (vehicle of hormone administered) birds. Treatment with melatonin, however, induced hyperglycemia in both NP and LP bird groups, _but hypoglycemia in SP birds. Unlike glycemic levels, amount of epinephrine (E) and norepinephrine ( E) in adrenals of control birds exhibited significant changes under altered photopcriods. A decrease in E and an increase in NE were noted in adrenals of both LP and SP birds. Exogenous melatonin in NP birds also caused a decrease in E and concomittant ri se in NE level s. On the other hand, treatment of melatonin in both LP and SP bird groups resulted in an increase in the qu antity of both E and NE compared to respective values in adrenals of melatonin injected NP birds. However, rel ative to the amount of E and NE in adrenals of placebo treated LP and SP birds, signifi cant effect of melatonin treatment was observed only in SP birds. The results suggest that influences of exogenous melatonin on the levels of both blood glucose and adrenal catecholami nes are largely modulated by short rather than long photoperiods.

The pineal gland acts as a neuroendocrine transducer which exhibits well-defined diurnal melatonin output as a response to photic stimuli and thus influences a number of metabolic and endocrine functions 1

'2

. The role of pineal gland and its hormone melatonin in the regulation of carbohydrate metabolism has been studied in mammals3

-5

, as well as in a few species of birds6

, including pigeons7-10 and roseringed

parakeets 11'12

. Attempts have also been made to demonstrate the influence of the pineal gland and/or its hormone melatonin on the functions of avian adrenal medulla 13

'14

. However, any functional relationship between the levels of adrenal catecholamines and blood glucose in the melatonin treated birds is as yet a topic of speculation . Likewise, despite recognition of the pineal organ as an intermediary between the environment and endocrine system in birds 15

, it was largely unexplored whether effects of melatonin on the levels of blood glucose and adrenal catecholamines are modulated by the changes in photoperiod. Accordingly, it was envisaged in the present investigation to demonstrate the glycemic and adrenal catecholaminergic responses to exogenous melatonin in adult male roseringed parakeets under natural and altered photoperiods.

Materials and Methods Collectioll atld mailltetlance of birds- Adult male

roseringed parakeets (Psittacula krameri Neumann; Psittaciformes; Aves) were collected locally (Lat. 23° 14'N; Long. 87°51'E), during February (natural photoperiod: around 11 hr 30 min) corresponding to the breeding phase in an annual test icular cycle 16

. The birds were acclimatized for about a week in an open air aviary under natural photoperiods, and 20-45% RH, and were provided with paddy (Oryza) as food and water ad libitum during acclimatization and throughout the experiment.

Experimelltal paradigm- A total of 36 birds weighing between 110-140 g were divided at random into three groups. Each group, containing 12 birds, was separately held under natural (NP), or long (LP; 16L:8D; mtificial lights were available in between 0600 and 2200 hrs) , or short (SP; 8L: 16D; artifici al lights were available in between 0600 and 1400 hrs) photoperiods. NP birds were maintained in an open­air aviary under natural conditions including the photoperiods which varied between 11 hr 30 min (at the onset of experiment) and 12 hr (at the end of experiment), though supplementary mtificial lights

1112 IND IAN J EXP BIOL, NOVEMBER 2000

were avai lable in the shaded areas of the aviary between 0800 and 1700 hr. In a ll cases artificial li ghts were provided with white fluorescent lamps (intensity of li ght varied between 250-300 lu x at cage level). The birds of ei ther photoperiodic group were subsequent ly divided into two sub-groups each containing 6 birds. The first sub-group received only the placebo solution (sa line-ethanol mixture in 9:1 v/v ratio) and were considered as the control for the melatonin treatment. T he birds belonging to the other sub-group, under each photoperiod ic schedule, received un. InJection of melatonin (S igma Chemica ls, USA) at the dose of 25 )lg/ 100 g body wt./ day for 30 consecutive days. This dose schedule of me latonin has been used by many investigators to study the 'melatoni n effect' in mammals 17 and is sti ll used in the study on birds 18

'19

. Though the influence of me laton in on avian gonad has been reported to be independent of the time of administration20

, generally exogenous administration of melatonin is done just before the onset of darkness in a 24 hr cycle, as this sc hedule has shown maximum efficacy of the treatments in several mammalian studies~ 1 •23 . Accordingly, the parakeets belonging to each photoperiodic group were injected daily with melatonin or its vehicle at the end of respective light phase.

Collection of blood samples and adrenals- On the following day of the last treatment, the blood sample

(200 )ll) from each bird was collected separately from the brachial vein during mid-noon, i.e. at around 12.00 noon when blood glucose level reaches its peak in parakeets 12

• Subsequently, the birds were sacrificed by cervical di slocation, and quick dissect ion was followed for the collection of both the adrenal glands from each indi vidual. After removal of extraneous ti ssues, the paired adrenals were soaked with blotting paper, weighed in a balance sensitive to 0.01 mg, and transferred to pre-chilled tubes containing 6% TCA solution .

Measurement of blood glucose levels- Quantity of g lucose in blood was estimated by g lucose oxidase­peroxidase method using ortho-di anisidine as the colour reagent24

. The value was expressed as mg of glucose/ dl of blood.

Measurement of concentrations of adrenal catecholamines- The adrenals were homogenized in 6% TCA, purified25 for quantitative determination of the amounts of epinephrine (E) and norepinephrine (NE) according to a method described elsewhere14

'26-28

.

The concentrations of E and NE in the adrenals were expressed as )lg/mg of tissue.

Analysis of data- The data were ana lysed by two­way analysis of variance CANOVA) fo llowed by two­tailed Student's t test29

. The criterion for significance was ?~0.05.

Results A. Influences of altered photoperiods on the

concentrations of blood glucose and adrenal catecholamines in control birds- The mean va lues of the concentrations of blood glucose, adrenal E and adrena l NE in di fferent bird groups have been presented in F igs. 1 and 2 respectively. The blood glucose level in control (vehicle of meliltonin injected) birds did not vary significantly with regard to the changes in photoperiods (Fig. 1). However, altered photoperiods (both LP and SP) induced a significant decrease in the concentration of E (Fig. 2A) and a concomitant rise in the level of NE (Fig. 2B) compared to the va lues in the ad renals of NP birds .

B. Effects of melatonin on the concentrations of blood glucose:

( i) Under natural photoperiodic conditions­Daily administrations of melaton in to NP birds resulted in hype rglycemia (Fig. l) compared to the blood glucose values in control birds.

( ii ) Under altered photoperiodic conditions­Glycemic response in parakeets to exogenous melatonin

170 [Sj CONTROL UIIlJ MELATONIN ADM INISTERED ., 0

"' 160 0

"" .s Vl

150 ..... w

i > 140 w ..... w 130 Vl 0 u 120 ::::> .....

"' 0 110 0 0

1: [ a ~ ~ ..... <D

NP LP SP

Fig. I -Diagrammatic presentation of the values (means±S.E.'s in vertical bars) of blood glucose level in natural (NP; about I 2L : 12D), long (LP; 16L: 8D), and short (SP; 8L: I6D) photoperiodic male roseringed parakeets foll owing daily administration of vehicle of hormone (control ) or melatonin for 30 consecutive days. ["Significant at 5% level, compared to control group under identical photoperiodic condition ; bSigniticant at 5% level. compared to corresponding group under natural photoperiodic condition.]

MAITRA eta/.: GLYCEMIC & CATECHOLAM!NERG IC RESPONSES TO MELATONIN AND PHOTOPERIODS 111 3

,,r 0 lSJ CONTROL b

600 IIIIlll MELATONIN ADMINISTERED I Vl -' UJ > UJ 400 -'

UJ -z "' oc E

b I o b "- <;? 300 w, ~ "' "- ::1.. UJ - b -' 200 <: z UJ 0: 0 <:

100

0 NP LP SP

900 ® b 0

b Vl

800 b --' -UJ > UJ

700 -'

UJ

~ 600 a:-I "' "- E 500 ~~ "-'-UJ "'

400 0 0: ::1.

d 0-

300 z -' <: 200 z UJ 0:

100 0 <:

0 NP LP SP

Fig. 2- Diagrammatic presentation of the values (means± S.E.'s in vertical bars) of the concentrations of epinephrine (A) and norepinephrine (B) in the adrenals of natural (NP), long (LP), and short (SP) photoperi odic groups following daily administration of vehicle of hormone (control ) or melatonin for 30 consecuti ve days. ["Significant at 5% level, compared to control group under identical photoperiodic condition ; bSignificant at 5% level , compared to corresponding group under natural photoperiodic condit ion.]

varied significantly in relation to photoperiodic conditions. While daily treatment of melatonin to LP birds resulted in hyperglycemia, a significant decrease in the values of blood glucose was noted in similarly treated SP birds relative to glycemic values m respective groups of control birds (Fig. 1).

C. Effects of melatonin on the concentrations of adrenal catecholanzines:

(i) Under natural photoperiodic conditions­Exogenous melatonin induced a significant reduction

in the concentration of E (Fig. 2A) and a concomitant increase in the level of NE (Fig. 2B) in the adrenals of birds maintained under natural photoperiodic conditions.

(ii) Under altered photoperiodic conditions­Compared to the values in plecbo (control) group of LP birds, no significant change was observed in the levels of adrenal E (Fig. 2A) and NE (Fig. 2B) in the melatonin treated LP birds. However, the concentrations of both E and NE were found to be significantly higher in the adrenals of LP birds than those measured with the adrenals of melatonin treated NP birds. On the other hand , a significant increase in the concentrations of both E (Fig. 2A) and NE (Fig. 2B) was noted in the adrenals of melatonin administered SP birds compared to the concentrations of respective adrenal catecholamines in control NP as well as in control SP groups of parakeets.

Discussion In mammals, light information is transferred from

the eyes to the pineal where it controls the production of melatonin30

. This idea has an implication that the lighting environment may play a role in the regulation of endogenous level of melatonin. Although this pathway is not yet clear in avian species, Liou31 and Liou et al32

. implicated both the light-dark cycle and melatonin in the entrainment of oviposition suggesting that the role of light and melatonin in birds is similar to that in mammals.

The results of the present study revealed that under natural photoperiodic conditions, the treatment of melatonin in parakeets results in hyperglycemia. Previous studies on pigeons following a single intravenous injection 7 or subcutaneous implantation for a 12-week period 10 of melatonin also demonstrated similar effect on the level of blood glucose. However, reports are available to show that glycemic response to melatonin administration may vary in relation to the time period during a circadian cycle7 or an annual reproductive cycle8

'9 at which melatonin is injected,

and also the dose of administered melatonin 12• In

consideration to the data in the literature and the observation made in this study, it is not surprising that the treatment of melatonin in male roseringed parakeets during the inactive phase in an annual gonadal cycle resulted in hypoglycemia 11

, and an identical treatment in the sexually active birds of the samr species caused hyperglycemia (present study). The etfects of pinealectomy on the blood glucose leve18

1114 INDIAN J EXP BIOL, NOVEMBER 2000

and on the metabolism of carbohydrate9 were also shown to vary in relation to the reproductive season in an annual cycle. These observations were taken to indicate a possible anti-insulinic/anti-avian pancreatic polypeptide role for the pinea l during the breeding phase and reverse actions of the pineal principle during the non-breeding phase in an annual gonadal cycle in wild pigeons. However, at present it is difficult to confirm a synergistic or antagonistic interac tion between the pineal and pancreas in relation to the breeding activity of roseringed parakeets .

The role of photoperiods on the regulation of gonadal functions has been well studied in several avian species, including the roseringed parakeets33

.

Under natural photoperiodic conditions recovery of gametogenes is from the prolonged resting phase in an annual testicular cycle in parakeets occurs after experiencing autumnal equinox, but active gametogenesis in the testis takes place only after winter solstice (corresponding to photoperiods of about 10 hrs 30 min) under the influence of increasing photoperiods ranging between 11 and 12 hr33

. A positive correlation has also been noted between the rate of testicular growth in free-living parakeets and the duration of environmental photoperiods in annual cycle 16

. However, the authors are not aware of any previous study on the influence of altered photoperiods on the glycemic leve l in any bird. It is ev ident from the results of the present study that neither LP nor SP has any significant effect on the concentration of blood glucose in control (veh icle of melatonin injected) parakeets. The glycemic response to melatonin was also identical among the NP and LP birds. But the level of blood glucose in melatonin administered SP birds was found to be significantly reduced compared to the glycemic values in both the control SP and melatonin injected NP parakeets. The results suggest that the duration of light as such may not have any influence on the blood sugar level in parakeets, but glycemic effect of melatonin is essentially dependent on the photoperiodic conditions under which the birds are held . In all probability, a short photoperiodic schedule may act as a modulator of the physiological responsiveness mechanism to the regulation of blood glucose level in the melatonin challenged bird. It may be noted here that exposure of hamsters to SP was also found to modify the effects of melatonin on the gonad34

.

Administration of melatonin in NP parakeets resulted in a significant decrease in the amount of

epinephrine (E) and a concomitant increase in the level of norepinephrine (NE) in the adrenals. Earlier studies on different mammalian species have shown that the pineal organ play an important role in the regulation of adrenal catecholaminergic functions 5

.

Admjnistration of melatonin may cause an increase as well as a decrease in secretory activity depending on the cell types of adrenal medulla35

. A study on the adrenal medulla of different species of birds following 0 .5 hr of a single intraperitonial injection of melatonin (0.5 mg/ LOO g body wt.) revealed a species specific and age specific response of E and NE cell 13

. The quantitative estimations of catecholamines in the adrenals of pinea lectomized and/or melatonin treated young chicks also demonstrated diffe rential behaviour of E and NE in the adrenals of concerned birds 14

. The biosynthesis of catecholamines in an avian adrenal medulla proceeds, probably following the mammalian pattern, from tyrosine to dopa to dopamine to norepinephrine to epinephrine36

. The conversion of NE to E requires methylation of NE by the enzyme phenylethanolamine-N-methyl transferase or PNMT37

.

In light of the existing knowledge, it may not be unwise to speculate that the increase in the amount of NE accompanied by the stoichiometric decrease of E in the adrenals of melatonin treated birds may result from: (a) an increased synthesis of NE by melatonin probably through inducing ACTH release, that in turn ac tivated tyrosine hydroxylase directl / 8

· 39 and/or (b)

a di rect or indirect inhibition of the activity of PNMT.

Unlike the levels of blood glucose, the concentrations of adrenal E and NE in different groups of control bird were found to undergo significant changes in relation to the schedule of photoperiod. Exposure of birds to both LP and SP schedules evoke an identical response in the level of adrenal catecholamines. In either case, a significant decrease in the concentration of E was accompanied by a concomitant increase in the level of NE compared to the respective values in the adrenals of NP birds. Although the functional etiology of the photoperiodic response of adrenal catecholamines remains unknown, the study on rats under continuous illumination and darkness also revealed identical pattern of synthesis and metabolism of adrenomedullary hormones40

. It has been suggested that one-month continuous exposure of rats to either light or darkness may lead to an appreciable decrease in the amount of E and the E synthesizing enzyme (PNMT) in the adrenals. There is a possibility that an altered

MAITRA eta/.: GLYCEM IC & CATECHOLAM INERGIC RESPONSES TO MELATONIN AND PHOTOPER IODS 1115

photoperiodic schedule modifies the tone of the sympathetic nervous system41

'42 related to the

secretion of catecholamines by the adrenal gland. The data on the adrenal catecholamines in the

melatonin administered altered photoperiodic birds are interesting. The concentrations o f adrenal E and NE in melatonin treated LP birds were not significantly different from those in the control birds held under LP schedule. On the other hand , the values of both E and NE in the adrenals of melatonin injected SP birds were found to be significantl y increased over the concentrations of respect ive adrenal hormones in control SP as well as melatonin treated NP birds. This variability between the influences of LP and SP on the glycemic and catecholaminergic effects of melatonin in parakeets may be explained as due to di ffe rence in the duration of melatonin to act to elicit its physiological response43

. In fowl prolonged darkness is known to modulate the endogenous mjlieu of melatonin31

.

Collectively, the results of thi s study indicates that the light/dark ratio influences e ither directly or indirectly the endocrine system44 involved in the regulation of adrenal catecholaminergic as well as glycemic responses to exogenous melatonin. In this respect, probably SP schedule is more effective modulator of melatonin effect than the LP schedule. The conjecture earns support from the results of earlier workers who have demonstrated that the synthesis of melatonin becomes significantly higher in the pineal of hamsters housed under short photoperiods45 and melatonin effects on the reproductive system is modified to larger extent by SP than LP in seasonally breeding mammals46

.

Nonetheless, the topic should remain speculative until measurement of melatonin titre in the currently considered avian species under given experimental conditions.

Acknowledgement The financial assistance from the

Science and Technology, Govt. acknowledged.

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