BIOLOGY OF REPRODUCTION 39,9-18 (1988)

Pineal and Ovarian Response to 22- and 24-h Days in the Ewe'

J. ENGLISH,3 J. ARENDT,2s A. M. SYMONS,3 A. L. POULTON,3 and I. TOBLER4

Department of Bio chemistry3 University o f Surrey

Guildford Surrey, England G U2 5XH

and Institute of Pharmacology4

University of Zurich 8006 Zurich, Switzerland

ABSTRACT Melatonin secretion in ewes was entrained by 22-h light-dark cycles whether of long (16L:6D) or short

(6L:16D) photoperiod. In photoperiods of 6L:16D, a phase-delay of melatonin secretion was evident, leading to a dark-phase duration shorter than that found in 8L:16D. Early onset of estrus was induced in anestrous ewes kept in 8L:16D, but not 6L:16D, f rom 22 July compared to controls in natural light. In photoperiods of 16L:6D, the melatonin profile corresponded precisely to the dark phase. Early offset of estrus was induced in estrous ewes kept in both 18L:6D and 16L:6D from 18 December compared to controls in natural light.

Thus, when the duration of melatonin secretion was appropriate to the long photoperiod (16L:6D), but with a constantly changing phase position, a long-day reproductive response was found. Activity-rest cycles were not entrained b y 16L:6D; thus the synchronization of melatonin and activityrest cycles does not appear to be essential for the induction of a long-day reproductive response.

These results support the hypothesis that the duration, not the circadian-phase position, of melatonin is critical to the induction of photoperiodic effects.

INTRODUCTION

A functional pineal gland is essential for the perception of changing daylength in a number of photoperiodic species (Reiter, 1980; Tamarkin et al., 1985 ; Arendt, 1986). The pineal hormone, melatonin, appears to be the photoperiodic messenger molecule. Removal of the daily melatonin rhythm by pinealec- tomy (Bittman et al., 1983) or by sympathetic denervation of the gland (Lincoln, 1979) abolishes the appropriate seasonal response to applied artificial photoperiod. Suitable administration of melatonin in physiological quantities to pinealectomized animals replicates the effects of varying photoperiod (Carter and Goldman, 1983a, b; Bittman and Karsch, 1984; Bittman, 1985). Essentially, the pineal gland, through

Accepted February 10, 1988. Received December 3, 1987.

Supported by the AFRC, U . K. Part of this work was presented at the 1. N. R.A. Colloquium on Photoperiodism, Nouzilly, France, September, 1987.

Reprint requests.

melatonin, appears to act as a synchronizer of seasonal cycles (Herbert, 1981; Hoffmann, 1987). How it achieves this is at present under intense investigation.

Melatonin is secreted at night: the rhythm is endogenous and both entrained and suppressed by light (Lincoln et al., 1985). In ewes, as indeed in a number of other species, the duration of nighttime secretion reflects the length of the dark phase (Arendt, 1985), and there is strong evidence, in ewes and hamsters, that the duration of melatonin secretion is the critical parameter in the induction of photoperi- odic responses (Carter and Goldman, 1983a,b; Gold- man, 1983 ; Bittman and Karsch, 1984). Evidence also exists, however, that the effects of melatonin depend on an underlying daily rhythm of sensitivity to the hormone (Stetson,and Tay, 1983 ; Whatson-Whitmyre and Stetson, 1983). One approach to the investiga- tion of these various possibilities in ewes would be the administration of melatonin in physiological quantities, with an appropriate duration, at different phases of the circadian cycle, and with different periodicities, in pinealectomized animals. An alterna- tive possibility would be to manipulate the light-dark

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10 ENGLISH ET AL.

cycle such as to generate endogenous melatonin at different circadian-phase positions. We have investi- gated the ability of short and long artificial photo- period to entrain melatonin secretion. At the same time, we have observed the accompanying ovarian response, together with some activity-rest cycles.

MATERIALS AND METHODS

Animals

Twenty-four intact, maiden, Suffolk-cross ewes, aged 1% to 2 yr, were used in these experiments. They were raised from birth in natural light prior to artificial photoperiod, and fed daily in the morning (1000-1100 h) on 1 kg ewe and lamb nuts (B. P. Nutrition Ltd., Witham, Essex, U. K.) and in the afternoon with 1 kg each crushed oats, flaked maize, and hay with water ad libitum. During artifi- cial photoperiod treatment, they were maintained in groups of 4 in separate light-controlled rooms. The rooms were cleaned only during the light phase of any photoperiod. The rooms were not temperature- controlled or sound-proofed. Light intensity at sheep's eye level was approximately 600 lux (cool, white, fluorescent light) during the light phase and less than 1 lux during the dark phase (25-watt red light bulbs).

Blood Sampling

Blood samples were drawn into Vacutainer tubes (Becton & Dickinson Ltd., Cowley, Oxfordshire, U. K.) with lithium-heparin as anticoagulant. Plasma was rapidly collected by centrifugation and stored at -20" prior to assay for melatonin and progesterone. Dark-phase blood samples were taken under dim red light (<1 lux).

Assays

Melatonin was assayed by direct radioimmunoassay (RIA) originally described by Fraser et al. (1983) and modified for use in ovine plasma by English et al. (1986). The antiserum was raised in sheep against N-acetyl-5-methoxytryptophan conjugated to thyro- globulin (Guildhay Antisera, Guildford, Surrey, U.K., antiserum no. G/S/704/8483). The major cross- reacting substances are N-acetyl tryptamine (0.97%), 6-hydroxymelatonin (0.38%) and N-acetyltrypto- phan (0.26%). Inter- and intraassay coefficients of variation on pools containing 24, 118, and 551 pg melatonin/ml were 8.9%, 5.3%, and 2.7%, and 8.6%,

7.6% and 2.3% respectively. The detection limit of the assay was 5 pg/ml. All the samples from one experiment on one sheep were measured in the same assay.

Progesterone

Reproductive status was assessed from measure- ments of progesterone concentration in plasma samples taken twice weekly throughout the experi- ments. Progesterone was assayed by an enzyme-linked immunoabsorbent assay adapted from one developed for use in heifers (Boland et al., 1985; Sauer et al., 1986) and described previously (English et al., 1986).

Activity Recording

Activity meters, described by Borbely ( 1984), were transported from Zurich to Guildford. Recording was initiated in Guildford, each meter being attached firmly to a collar around the ewe's neck. At the end of the recording period, the meters were returned to Zurich for read-out.

Control of Light-Dark Cycles

Each animal room was equipped with a program- mable light timer such that 7 days of data could be pre-programmed. The timers were accurate to within 2-3 min.

Statistics

The onset and offset of melatonin secretion for each ewe was defined as the first point and last point more than two standard deviations above basal values during the light phase, or the anticipated light phase. In practice, most basal values were undetectable (<5pg/ml), so that these points were usually consid- ered to be the first and last values more than twice the limit of detection of the assay. The duration of melatonin secretion was taken as the time elapsed between onset and offset of secretion.

The date of onset of estrus was taken to be the date of the first of two successive plasma samples with progesterone values above 1 ng/ml, providing that they were followed by two cycles each of which had two successive plasma samples with progesterone concentrations of above 1 ng/ml. Similarly, the end of estrus was defined as the last day on which plasma progesterone was above 1 ng/ml, assuming that it had been preceded by two cycles as described above.

PINEAL AND OVARIAN RESPONSE TO 22 AND 24 h DAYS IN THE EWE 11

Differences between treatments were analysed by Student's t-test, analysis of variance, and Dunnett's test for least-significant difference, as appropriate.

Experimental Design Experiment 1. Anestrus was verified by progester-

one assay in three groups of 4 ewes. They were then maintained in two different, short photo- periods-81:16D (A) and 6L:16D (B)-and in natu- ral light. (C) from 22 July 1985. Blood samples were taken twice weekly for progesterone measurement. After 66 days, when dark onset coincided in Groups A and B, blood was sampled from these groups every hour for 48 h under the prevailing photoperiod. After 121 days, again when dark onset coincided in these two groups, blood was sampled hourly for 48 hours in darkness throughout. Samples were assayed for progesterone and melatonin.

Experiment 2. After 18 wk (25 November 1985) Experiment 1 was terminated and the light-dark cycles were reprogrammed to long photoperiod, such that Group A then received 18L:6D and Group B received 16L:6D. Group C remained in natural light. Blood was sampled twice weekly for progesterone measurement. After 77 days, when dark onset coin- cided in the two artificial photoperiod groups, blood was sampled hourly from these groups for 48 h under the prevailing photoperiod. After 132 days, again when dark onset coincided in the two groups, blood was sampled hourly for 48 h in complete darkness. This experiment was terminated on 6 April 1986 after 19 wk. Samples were assayed for progesterone and melatonin.

Experiment 3. In October 1986, three new groups of 4 ewes were maintained in natural light for 9 wk. Blood was sampled weekly for progesterone to determine reproductive status prior to experimenta- tion.

On 18 December, one group (D) was placed in an artificial photoperiod of 18L:6D and another group (E) in 16L:6D. Group F remained in natural light. Blood was sampled twice weekly thereafter for progesterone measurement. After 60 days when dark onset was 180" out of phase in the two groups D and E, blood was sampled hourly from these groups for 48 h in the prevailing photoperiod. Samples were assayed for progesterone and melatonin.

Activity-rest cycles were monitored continuously for 21 days from 21 January (i.e. from 34 days after the beginning of treatment), thus avoiding the days of

hourly blood sampling. The experiment was termin- ated on 14 April 1987, and the ewes were maintained in natural light until 9 June when they were again placed in 18L:6D (Group D) and 16L:6D (Group E). Beginning on 2 4 June, i.e. 15 days after transfer to artificial photoperiod, activity-rest cycles were again monitored-this time for 16 days.

R ESU LTS

Experiment 1 Progesterone. Prior to the start of the experiment,

all ewes were in anestrus with progesterone levels < 1 ng/ml. Estrus cycles began in Group A (8L: 16D) 43 * 1 day, in Group B (6L:16D) 71 f 5 days, and in

EXPERIMENT 1

ESTRUS ONSET

I

6L:16D

I Julv I Auaust FIG. 1 . Presence of estrous cycles (burs) in three groups each of 4

intact ewes kept in natural photoperiod or in short days of 8L:16D (24-h day) or 6L:16D (22-h day) from 22 July. The date of onset of estrus was taken to be the date of the first of two successive plasma samples to give a progesterone value above 1 ng/ml, providing that this was followed by two cycles-each of which had two successive plasma samples with progesterone concentrations above 1 ng/ml. Progesterone levels were assessed twice weekly. Estrus onset (days after 22 July was as follows: natural photoperiod, 58 f 2; 8L:16D, 43 f 1; 6L:16D, 71 k 5, mean f SEM. Estrus onset was earlier in 8L:16D compared to natural photoperiod (p<O.OOl), but the effect of 6L:16D was indistin- guishable from natural photoperiod.

12 ENGLISH ET AL.

Group C (natural light) 58 f 2 days (X f SEM) after the start of the experiment. Estrus onset in Group A was significantly earlier than in Group C (p<O.OOl). The time of estrus onset in Groups B and C was indistinguishable, although Group B was somewhat late (Fig. 1).

Melatonin. Melatonin profiles in Group A (8L: 16D) on Days 66 and 67 after onset of treatment corres- ponded to previous findings, i.e. a nighttime rise in all animals, with a duration corresponding to the length of the dark phase (Fig. 2). In Group B (6L:16D), the onset of melatonin secretion was 6 h after dark onset on Day 66 and 8 h after dark onset on Day 67, terminating in each case at light onset. There was thus a phase-delay in the onset of melatonin production, leading to a duration of secretion shorter than that of the prevailing dark phase (10 f 1 h, on Day 66, and 9 f 1 h on Day 67, X f SEM). The duration of mela- tonin secretion was significantly shorter in Group B than in Group A (t-test, p<O.Ol Day 66; p<O.OOS Day 67). The corresponding figures for Group A were 14 f 1 h for both days.

The melatonin profiles obtained during 48 h of darkness 121 days after onset of treatment are shown in Figure 3 . The profile corresponded to the antici- pated dark phase in Group A as expected. In Group B, onset of secretion was delayed with respect to the anticipated dark phase by 7 h on Day 121 and by 9 h on Day 122.

Experiment 2

Progesterone. Progesterone profiles for Experiment 2 indicated that ewes in Group A (18L:6D) ceased ovarian cycles 79 f 7 days after the start of this experiment, whereas Group B (16L:6D) ceased after 116 f 5 days. The two groups were not comparable because of their different reproductive histories at the beginning of the experiment, and no conclusions will be drawn from this part of Experiment 2. The effect of long photoperiod given as 22- or 24-h cycles on reproductive function was properly assessed in Experiment 3 .

Melatonin. Melatonin profiles in Group A (18L:6D) on Days 77 and 78 after the start of the experiment

EXPERIMENT 1

300

200

E n \ En

5 100

DAY 66

‘ I \

I, I I

DAY 67

8L: 16D _ _ _ - 6L16D

I j 8 L : l 6 D 6L: 16D

12 18 24 06 12 18 24 06

clock time

FIG. 2 . Plasma melatonin profiles (mean pg/rnl t SEM) in two groups of 4 ewes maintained for 66 days o n short-day photoperiods of 8L:16D (24-h day) and 6L:16D (22-h day) and sampled hourly for 48 h in the ambient photoperiod. The onset of dark-phase melatonin secretion is delayed 6-8 h in 6L:16D. Dark burs represent the dark phase.

PINEAL AND OVARIAN RESPONSE TO 22 AND 24 h DAYS IN THE EWE

EXPERIMENT 1

13

DAY 121 DAY 122

t 8L:16D ' 6L16D

12 18 24 06 12 18 24 06 clock time

FIG. 3. Plasma melatonin profiles (mean pg/rnl * SEM) in two groups of 4 ewes maintained for 121 days on short-day photoperiods of 8L:16D , * A I~ I -~, I I - .*- I^^. 3 . . . . . . * _ _ . . . . . " . . . . . _. . . . . . . . i ~ 4 - n aayj ana OL:IOU (LL-n aay) and sampled nourly tor 48 n, witn continuous darkness trorn 1400 h . l h e anticipated dark phase is represented by the dark burs and the anticipated light phase by hatched bars. Melatonin secretion corresponds to the anticipated dark phase after 8L:16D, but is delayed by 7-9 h after 6L:16D.

again corresponded to previous observations, i.e. a short duration of high levels corresponding precisely to the dark phase (Fig. 4). In Group B (16L:6D), in contrast to the 22-h cycle of 6L:16D employed previously, melatonin secretion also precisely followed the dark phase. There was no delay in the onset of secretion at night. Thus a 22-h cycle with a long light phase was able to entrain melatonin secretion with no apparent phase-delay.

When melatonin was determined during 48 h of darkness following 132 days of 18L:6D (A) or 16L:6D (B), a dramatic difference was seen (Fig. 5) compared to the profiles obtained in the prevailing photoperiod (Fig. 4). Both groups reverted to a long-duration secretion pattern-Group A: 10 f 2 h on Day 132 and 11 f 2 h onDay 133;Group B: 11 f 2 h on Day 132 and 14 f 2 h on Day 133. In both groups, onset of secretion closely corresponded to the anticipated dark phase on both Day 132 and Day 1 3 3. Considerable variability in the profiles was present, as indicated by the very large standard errors.

Experiment 3

Progesterone. The progesterone profiles for ewes in Experiment 3 are shown in Figure 6. Prior to artificial photoperiod treatment from 18 December, all ewes had undergone at least 2 ovarian cycles. Groups D (18L:6D), E (16:6D), and F (natural light) ceased ovarian cycles on Days 60 f 3, 59 f 7, and 85 f 5, J I * SEM, respectively, after the start of the experiment. Groups D and E both stopped cycling significantly earlier than Group F. (Group D t-test, p<O.Ol, Group E, p<0.05). The three groups were subjected to one-way analysis of variance ( p = 0.0101), followed by Dunnett's test for least-significant difference, again indicating early estrus-offset in Groups D and E ( p = 0.5).

Melatonin. Melatonin profiles in Groups D and E 60 days after the start of this experiment corresponded precisely to the dark phase (Fig. 7). It appears, therefore, that 22-h cycles of 16L:6D are able to entrain melatonin secretion completely. High levels of melatonin correspond to the dark phase whether

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EXPERIMENT 2

DAY 78

24 06 12 18 24 06 12 18

clock time

FIG. 4. Plasma melatonin profiles (mean pg/ml f SEM) in two groups of 4 ewes maintained for 77 days on long-day photoperiods of 18L:6D (24-h day) and 16L:6D (22-h day) and sampled hourly for 48 h in the ambient photoperiod. The dark phase is represented by the dark bars. Mela- tonin secretion corresponds to the dark phase in both groups and appears to be entrained with no phase-delay in 16L:6D.

positioned during the natural night or during daytime, as in this experiment (compare Experiment 2, Fig. 4), and whether or not the dark phase is synchronous with that of sheep on a 24-h cycle in an adjacent pen.

Activity recording. The activity records from 6 of the 8 sheep in Experiment 3 during the first recording session beginning 34 days after transfer to artificial photoperiod in January are shown in Figure 8. Two records from Group E (16L:6D) were lost due to battery failure. With only two sets of data from this group, it is impossible to draw any firm conclusions. The data are nevertheless of interest. A 24-h activity- rest cycle (dark portions represent activity) was clearly present in 3 of the 4 sheep in Group D (18L: 6D). The fourth sheep was only weakly rhythmic, yet a 24-h component was clearly present. Neither of the 2 sheep in Group E (16L:6D) showed any evidence of a 22-h rhythmic component in activity-rest. A second set of activity recordings were obtained from the same sheep kept in the same photoperiods over a period of 16 days beginning 15 days after the onset of treatment in June. On this occasion, a fragmented

24-h rhythm was seen in all sheep, with activity corresponding to feeding times (not shown). There was no evidence of a 22-h rhythm in Group E. Thus it would appear that although a 16L:6D cycle is able to entrain melatonin, it is probably outside the range of entrainment of the activity-rest cycle in sheep main- tained from the point of view of feeding, tempera- ture, and noise variation on a 24-h day.

DISCUSSION

The melatonin rhythm appeared to entrained by 22-h cycles whether of 6L:16D or 16L:6D. A phase- delay in the melatonin rise was evident in the 6L: 16D photoperiod, as well as in continuous darkness, with respect to the anticipated dark phase, but it was not completely consistent over the two cycles studied. In spite of these problems, it is nevertheless likely that entrainment occurred, with a persistent phase-delay, throughout the experiment. Such a phase delay is characteristic of systems where a driving force (Zeit- geber), in this case the light-dark cycle, entrains a

PINEAL AND OVARIAN RESPONSE TO 22 AND 24 h DAYS IN THE EWE

EXPERIMENT 2

I DAY 132 DAY 133 18L:6D

- - _ _ - 16L:6D

15

1 I ,1111 M I I ,I ,,,,,I I , ' 1wL:6D

l6L:6D 24 06 12 18 24 06 12 18

clock time

FIG. 5 . Plasma melatonin profiles (mean pg/ml t SEM) in two groups of 4 ewes maintained for 132 days o n long-day photoperiods of 18L:6D (24-h day) and 16L:6D (22-h day) and sampled hourly for 48 h, with continuous darkness from 0200 h. The anticipated dark phase is represented by the dark bars and the anticipated light phase by hatched bars. Melatonin secretion extends well beyond the anticipated dark phase in both groups, with onset of secretion corresponding to the anticipated dark phase after 18L:6D but with some delay after 16L:6D. Considerable variability of melatonin levels was evident.

EXPERIMENT 3 ESTROUS TERMINATION

I natural photoperiod

1 1 , 18L6D

16L6D : r l Ja nuary I Feb ruarv I M arch

self-sustaining oscillation, in this case melatonin, of inherently lower frequency (Aschoff, 1979). This implies that animals perceived a melatonin profile shorter than the prevailing inductive dark phase and with a 22-h periodicity. The reproductive system did not respond with onset of early estrous cycles in this group of sheep (B) compared to a group (A) receiving an identical length of dark phase with a 24-h period, or compared to a group (C) of sheep in natural light. There are several possible explanations for the lack of response. The duration of secretion may have been

FIG. 6. Presence of estrous cycles (bard in three groups of 4 intact ewes kept in natural photoperiod or in long days of 18L:6D (24-h day) or 16L:6D (22-h day) from 18 December. Estrus offset was defined as the last day on which plasma progesterone was greater than 1 ng/ml. providing that this was preceded by two cycles, each of which had two successive plasma samples with progesterone above 1 ng/ml. Progester- one levels were assessed twice weekly. Estrus offset (days after 18 December) was as follows: natural photoperiod, 85 t 5 ; 18L:6D, 60 ? 3; 16L:6D, 59 ? 7, mean i: SEM. Both 18L:6D and 16L:6D groups ceased estrous cycles earlier than the natural photoperiod group (p<0.05, ANOVA & Dunnetts test for least-significant difference).

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DAY 60

ENGLISH ET AL.

EXPERIMENT 3

DAY 61 .- 18LSD ---- 16LSO

200

- E \ 0) P

5

100

I - 18LaD 16LSD

12 18 24 06 12 18 24 06

clock time

FIG. 7. Plasma melatonin profiles (mean pg/ml t SEM) in two groups of 4 ewes maintained for 60 days in long-day photoperiods of 18L:6D (24-h day) or 16L:6D (22-h day) and sampled hourly for 48 h in the ambient photoperiod, with the dark periods out of phase in the two groups. Melatonin secretion corresponds to the dark phase in both groups and is entrained with no phase-delay.

too short, the circadian-phase position may have been inappropriate, or these two phenomena may have combined to prevent a reproductive response. Another possibility can be envisaged in that should a circa- dian rhythm of sensitivity to melatonin exist it may have been disrupted by this particular photoperiodic manipulation.

Melatonin was clearly entrained by 22-h cycles of 16L:6D, with no phase-delay and with an immediate decompression of the rhythm upon transfer to continuous darkness. The change in phase-relationship between the melatonin rhythm and the 1ight:dark cycle seen using these two different photoperiods is consistent with previous observations that the phase of a driven rhythm is dependent on photoperiod history (Pittendrigh, 198 1).

To our knowledge, this is the first observation of entrainment of melatonin rhythms to such short cycles, although Lincoln et al. (1985) have previously shown entrainment to 23-h and 25-h cyclesof 16L:7D and 16L:9D.

The entrainment of melatonin by 16L:6D provided an opportunity to generate a duration of melatonin secretion, appropriate to the photoperiod but with a constantly changing phase-position relative to the

24-h day. Ewes in Experiment 3 responded to this treatment (16L:6D) in the same way as those in 16L:8D (24-h cycle), i.e. by early arrest of estrous cycles. The small number of animals used in these experiments indicates that caution must be used in the interpretation of the results. The simple explana- tion of these observations is nevertheless that the duration, not the phase-position of melatonin, is vital for the appropriate reproductive response. This is entirely consistent with previous observations in hamsters (Goldman, 1983) and sheep (Bittman, 1985). The question as to whether a circadian rhythm of melatonin sensitivity exists is not entirely resolved, however. Should this rhythm of sensitivity exist in sheep, it is likely to display the entrainment characteristics of other circadian rhythms. It may be entrained by 22-h 1ight:dark cycles. I t may even be entrained by melatonin itself. At present, we can only speculate as to whether the manipulations reported here are likely to have entrained a rhythm of mela- tonin sensitivity. To this end, the recording of activity- rest cycles, considered as a representative circadian rhythm, was of particular interest. Very little infor- mation is available on sheep rest-activity cycles, although a free-running rhythm has been described

PINEAL AND OVARIAN RESPONSE TO 22 AND 24 h DAYS IN THE EWE 17

18L6D ACTIVITY RECORDS a

11 17 23 'T' . 1 1

- 11 17 23 05 * I 1 w-

FIG. 8. (a) Activity records of 4 ewes maintained in long-day photo- periods of 18L:6D (24-h day). Activity recording was initiated after 34 days in this photoperiod. Successive daily records are shown for each ewe (1-4) with mean activity (arbitrary units) for each day displayed on the right and mean activity (arbitrary units) over the entire recording period as a function of the time of day is shown below each record. Ewes 1, 3, and 4 show clear 24-h variations in activity. Ewe 2 shows a weak activity rhythm, which nevertheless appears to have a 24-h periodicity. The dark phase is indicated by the enclosed urea. Clock time ( t ) is shown in hours (h) .

FIG. 8. (b) Activity records of 2 ewes maintained in long-day photoperiods of 16L:6D (22-h day). Recording was initiated after 34 days in this photoperiod and is displayed as in (a) Neither 24-h nor 22-h rhythms are apparent. The dark phase is indicated by the enclosed urea. Clock time ( t ) is shown in hours (h) .

t h 11 17 23 - 05

4

U t h

l W 1 -

ACTIVITY RECORDS b 16LeD

11 17 23 05 11 t h

11 17 23 05 11 t h

18 ENGLISH ET AL.

(Hunsaker and Wolynetz, 1979), and, using the same activity recording device, 24-hour rhythms with most activity during the day are always seen in a natural environment (Tobler, I. personal communication). The sheep recorded here in 16L:6D showed no 22-h rhythmicity in activity-rest, but nevertheless showed an entrained melatonin rhythm and gave an appro- priate photoperiodic response to 6 h of darkness perceived with 22-h periodicity. I t is thus possible that the coupling of the rhythms of melatonin and activity-rest reported in the hamster (Tamarkin et al., 1980) is not a prerequisite for a photoperiodic response in sheep. Even so, it is still possible that a hypothetical rhythm in sensitivity to melatonin may be entrained in 16L:6D in the absence of entrain- ment of activity-rest cycles. Other experiments in hamsters may suggest a dissociation between photo- periodic function and the suprachiasmatic nucleus, a central circadian rhythm generator (Hastings et al., 1985), in that neurotoxic lesions of the hypothala- mus were able to disrupt photoperiodic function whilst preserving the activity-rest cycle.

One final point may be made concerning the dissociation between activity-rest cycles and melatonin in the 16L:6D ewes. There has been much discussion and some evidence that melatonin can entrain activity- rest cycles in rodents (Redman et al., 1983). Whether or not it has such properties in ovines, it is clear that melatonin was not able to entrain activity-rest cycles to a 22-h periodicity in these experiments.

REFERENCES

Arendt J. 1985. Mammalian pineal rhythms. Pineal Res Rev 3:161-213 Arendt J, 1986. Role of the pineal gland and melatonin in seasonal

reproductive function in mammals. Oxford Reviews of Reproduc- tive Biology 8:266-320

Aschoff J. 1979. Circadian rhythms: general features and endocrinolog- ical aspects. In: Kreiger DT (ed.), Endocrine Rhythms. New York: Raven Press, pp. 1-61

Bittman EL, 1985. The role of rhythms in the response to melatonin. In: Evered D, Clark S (eds.), Photoperiodism, Melatonin and the Pineal Gland, Ciba Foundation Symposium. London: Pitman, pp. 149-69

Bittman EL, Dempsey J, Karsch FJ, 1983. Pineal melatonin secretion drives the reproductive response to daylength in the ewe. Endo- crinology 113:2276-83

Bittman EL, Karsch FJ, 1984. Nightly duration of pineal melatonin secretion determines the reproductive response to inhibitory daylength in the ewe. Biol Reprod 30:585-93

Boland MP, Foulkes JA, MacDonnell HF, Sauer MJ, 1985. Plasma progesterone concentrations in superovulated heifers determined by enzymeimmunoassay and radioimmunoassay. Br Vet J 141:

Borbely A, 1984. Ambulatory motor activity monitoring to study the rimecourse of hypnotic action. Br J Clin Pharmacol 18:835-65

Carter DS, Goldman BD, 1983a. Antigonadal effects of timed melato- nin infusion in pinealectomised male Djungarian hamsters (Pho- dopus sungorus sungorus): deviation is the critical parameter. Endocrinology 213:1261-67

Carter DS, Goldman BD, 1983b. Progonadal role of the pineal in the Djungarian hamster (Phodopus sungorus sungorus): mediation by melatonin. Endocrinology 113: 1268-73

English J , Poulton AL, Arendt J , Symons AM, 1986. A comparison of the efficiency of melatonin treatments in advancing oestrus in ewes. J Reprod Fertil 77:321-27

Fraser S , Cowen P, Franklin M. Franey C, Arendt J, 1983. Direct radioimmunoassay for melatonin in plasma. Clin Chem 29:

Goldman BD, 1983. The physiology of melatonin in mammals. Pineal Res Rev 1:145-82

Hastings MH, Roberts AC, Herbert J , 1985. Neurotoxic lesions of the anterior hypothalamus disrupt the photoperiodic but not the circadian system of the Syrian hamster. Neuroendocrinology 40:316

Herbert J , 1981. The pineal gland and photoperiodic control of the ferrets reproductive cycle. In: Follert BK, Follett DE (eds.), Biological Clocks in Seasonal Reproductive Cycles. Bristol: Wright,

Hoffmann K. 1987. The role of the pineal gland in the photoperiodic control of seasonal cycles in hamsters. In: Follett BK, Follett DE (eds.), Biological Clocks in Seasonal Reuroductive Cvcles. Bristol:

409-15

396-97

pp. 261--76

Wright, pp. 237-50 Hunsaker WK, Wolynetz MS, 1Y7Y. A study o t tree-running rnytnms in

sheep. Int J Chronobiol 9: 219-30 Lincoln GA, 1979. Photoperiodic control of seasonal breeding in the

ram: participation of the cranial sympathetic nervous system. J Endocrinol 82:135-47

Lincoln CA, Ebling FJP, Almeida OFX, 1985. Generation of melatonin rhythms. In: Ciba Foundation Symposium 117. Photoperiodism, Melatonin and the Pineal. London: Pitman, pp. 129-141

Pittendrigh CS, 198 1. Circadian organization and the photoperiodic phenomena. In: Follett BK, Follett DE (eds.), Biological Clocks in Seasonal Reproductive Cycles. Bristol:Wright, pp. 1-35

Redman J , Armstrong S, Ng KT. 1983. Free running activity rhythms in the rat: entrainment by melatonin. Science 219:1089-91

Reiter RJ, 1980. The pineal and its hormones in the control of repro- duction in mammals. Endocri Rev 1:109-31

Sauer MJ, Foulkes JA, Worsfold A, Morris BA, 1986. Use of proges- terone-1 1-glucuronide-alkaline phosphatase conjugate in a sensitive microtitre plate enzyme immunoassay of progesterone in milk and its application to pregnancy testing in cattle. J Reprod Fertil

Stetson MH, Tay DE, 1983. Time course of sensitivity of golden hamsters to melatonin injections throughout the day. Biol Reprod

Tamarkin L, Baird CJ, Almeida OFX, 1985. Melatonin: a coordinating signal for mammalian reproduction. Science 227:714-20

Tamarkin L, Reppert SM, Klein DC, Pratt B, Goldman BD, 1980. Studies on the daily pattern of pineal melatonin in the Syrian hamster. Endocrinology 107: 1525-29

Watson-Whitmyre M, Sterson MH, 1983. Simulation of peak melatonin release restores sensitivity to evening melatonin injections in pinealectomised hamsters. Endocrinology 112:763-65

76:375-91

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