Effects of prolonged artificial photoperiod on circulating prolactin and melatonin levels in seasonal ewes

Crossbred ewes exposed to long days for 46 months prior to photoperiod reversal showed no alteration in the duration or amplitude of the circulating melatonin peak between 24 and 46 months of continuous long day exposure. By 3 months after photoreversal to short days, both the amplitude and duration of the peak had adapted to the new scotophase. In short day treated ewes, the melatonin peak was abolished by 46 but not 24 months of short day exposure, and was not fully restored in all ewes 3 months after photoreversal. Mean prolactin levels over 24 h remained high up to 46 months of long day treatment, and declined 3 months after short day exposure. Conversely, mean prolactin levels remained low up to 46 months of short day treatment, increasing 3 months after exposure to long days. Thus: (i) depletion of the melatonin-synthesizing capability of the ovine pineal gland by prolonged exposure to long nights is not completely reversed after 3 months of continuous long day exposure, and (ii) a nocturnal melatonin peak is not essential for maintenance of plasma prolactin levels under these conditions.Special issue dedicated to Dr. Lawrence Austin.     

Prolactin response in Border-Leicester x merino ewes to administration of melatonin, melatonin analogues, a melatonin metabolite and 6-methoxybenzoxazolinone

The effect of structural modifications of the melatonin molecule on plasma half-life of the analogues and basal prolactin secretion was studied in Border-Leicester x Merino ewes. Halogenation at position 6 and/or unsaturation of the 2,3-double bond of the melatonin molecule slightly lengthened the half-life of the analogues. Melatonin, 6-chloromelatonin, 2,3-dihydromelatonin and 6-chloro-2,3-dihydromelatonin decreased plasma prolactin to 31, 45, 54 and 48% of control levels respectively when administered daily (100 micrograms at 1600 h) for 21 days. The brain metabolite of melatonin, N-acetyl-N'-formyl-5-methoxykynurenamine, and the putative natural melatonin analogue, 6-methoxybenzoxazolinone, failed to affect prolactin levels when administered in a similar manner. These results indicate that certain structural modifications to the melatonin molecule can be tolerated biologically; however, the modifications reported here still did not prevent rapid clearance from the circulation.     

Photoperiodic history and a changing melatonin pattern can determine the neuroendocrine response of the ewe to daylength

The reproductive neuroendocrine response of Suffolk ewes to the direction of daylength change was determined in animals which were ovariectomized and treated with constant release capsules of oestradiol. Two groups of animals were initially exposed to 16 or 10 h light/day for 74 days. On day zero of the study, when one group of ewes was reproductively stimulated (elevated LH concentrations) and the other reproductively inhibited (undetectable LH concentrations), half the animals from each group were transferred to an intermediate daylength of 13 h light/day. The remaining ewes were maintained on their respective solstice photoperiods to control for photorefractoriness. LH concentrations rose in animals experiencing a 3 h decrease in daylength from 16L:8D to 13L:11D while LH concentrations fell to undetectable values in those that experienced a 3 h increase in daylength from 10L:14D to 13L:11D. The photoperiodic response of the Suffolk ewe, therefore, depends on her daylength history. Such a result could be explained if the 24-h secretory pattern of melatonin secretion, known to transduce photoperiodic information to the reproductive axis, was influenced by the direction of change of daylength. Hourly samples for melatonin were collected for 24 h 17 days before and three times after transfer to 13L:11D. The melatonin secretory profile always conformed to daylength. Therefore, the mechanism by which the same photoperiod can produce opposite neuroendocrine responses must lie downstream from the pineal gland in the processing of the melatonin signal.     

Reproductive response and LH secretion in ewes treated with melatonin implants and induced to ovulate with the ram effect

Two experiments were conducted to examine the effects of treating seasonally anoestrous ewes with melatonin before ram introduction on reproductive response, and on LH secretion in anoestrous ewes induced to ovulate by rams.In Experiment 1, a total of 667 ewes from three flocks involving Merino (Flock 1, N = 149), Merino entrefino (Flock 2, N = 325) and Rasa Aragonesa (Flock 3, N = 203) breeds were used. Within each flock, ewes isolated from rams since the previous lambing were assigned at random to receive melatonin implants of Regulin (75, 175 and 105 in Merino, Merino entrefino and Rasa Aragonesa flocks, respectively) or to serve as untreated controls (74 in Merino, 150 in Merino entrefino and 98 in Rasa Aragonesa flocks). Fertile rams were introduced into all flocks 5 weeks after implantation in March (Flocks 1 and 2) or April (Flock 3), and remained with the ewes for a 50 day mating period. Percentage of ewes with luteal activity at ram introduction did not differ between melatonin treated and control ewes in any flock. There were no significant differences in either the mean interval from ram introduction to lambing or the distribution of lambing. Implantation with melatonin resulted in an improvement of prolificacy in all three flocks, although this only reached statistical significance in the Merino flock (1.15 vs. 1.03 in treated and control ewes, respectively, P < 0.05). Fertility was increased significantly (P < 0.05) in the Merino entrefino flock (64.5% in treated vs. 51.3% in control ewes).In Experiment 2, two trials were undertaken utilizing a total of 63 ewes. Trial 1 involved 24 mature Manchega ewes and Trial 2 involved 39 Merino ewe lambs. Half of the animals in each trial received a Regulin implant on 28 February (Trial 1) or 12 March (Trial 2) and the remaining half acted as controls. Rams were introduced 5 weeks after implantation and remained with the ewes for a 25 day period. In both trials, anoestrous ewes at ram introduction were bled at 20 min intervals for 3 h before and 5 h after ram introduction and then at 3 h intervals over the next 24 h for assessment of plasma concentrations of LH. Secretion of LH before or following introduction of rams was not affected by melatonin. Both treated and control anoestrous ewes in each trial responded to introduction of rams with an increase in the frequency of the LH pulses (P < 0.05), but no significant changes were detected in pulse amplitude or mean levels of LH. A preovulatory surge of LH was detected between 8 and 26 h after ram introduction, but neither mean interval from ram introduction to the peak of LH surge, nor the magnitude of the LH peak, was influenced by melatonin treatment.Results from this study show that: (1) melatonin implants administered during early seasonal anoestrus have the potential to improve reproductive performance in Spanish breeds of sheep, but the response is conditioned by breed, management system and environmental factors; (2) melatonin did not modify the secretion of LH in anoestrous ewes induced to ovulate by the ram effect under our experimental conditions.     

Effects of melatonin implants in ram lambs

Sixteen pinealectomized and 19 unoperated ewes were exposed to constant light for about 4 weeks before and 4 weeks after lambing. Six ram lambs born to unoperated ewes were implanted s.c. with melatonin sachets while 8 ram lambs were implanted with empty sachets. The 8 ram lambs born to pinealectomized dams also received empty implants. Ewes and lambs were then returned to the field. Analysis of weekly blood samples indicated that prolactin secretion was significantly decreased in the ram lambs with empty implants between 44-51 weeks of age whereas lambs treated with melatonin failed to show a significant change during development. All 3 groups of rams had elevated LH levels between 7 and 17 weeks of age, and a second period of high LH between 27 and 40 weeks. There were no significant differences between groups in the patterns of FSH secretion; FSH was highest between 7 and 17 weeks of age, and again between 27 and 40 weeks of age. Plasma testosterone levels in all groups increased gradually between 4 and 35 weeks. Between 38 and 48 weeks of age testosterone concentrations were markedly elevated in all groups. Growth was not affected by melatonin treatment. These results indicate that neonatal melatonin treatment has subtle endocrine effects; whether these effects are sufficient to compromise fertility in the ram, however, remains to be established.     

Daily and circadian variations of the pineal and ocular melatonin contents and their contributions to the circulating melatonin in the Japanese newt, Cynops pyrrhogaster

Daily and circadian variations of melatonin contents in the diencephalic region containing the pineal organ, the lateral eyes, and plasma were studied in a urodele amphibian, the Japanese newt (Cynops pyrrhogaster), to investigate the possible roles of melatonin in the circadian system. Melatonin levels in the pineal region and the lateral eyes exhibited daily variations with higher levels during the dark phase than during the light phase under a light-dark cycle of 12 h light and 12 h darkness (LD12:12). These rhythms persisted even under constant darkness but the phase of the rhythm was different from each other. Melatonin levels in the plasma also exhibited significant day-night changes with higher values at mid-dark than at mid-light under LD 12:12. The day-night changes in plasma melatonin levels were abolished in the pinealectomized (Px), ophthalmectomized (Ex), and Px+Ex newts but not in the sham-operated newts. These results indicate that in the Japanese newts, melatonin production in the pineal organ and the lateral eyes were regulated by both environmental light-dark cycles and endogenous circadian clocks, probably located in the pineal organ and the retina, respectively, and that both the pineal organ and the lateral eyes are required to maintain the daily variations of circulating melatonin levels.     

Effect of frontal hypothalamic deafferentation on duration of breeding season and melatonin secretion in the ewe

The objectives of this study were to determine if ewes subjected to frontal hypothalamic deafferentation (FHD) during anestrus remained anestrus or began to have estrous cycles, and if melatonin secretion was disrupted by FHD. Ovary-intact ewes in Group 1 were subjected to either FHD (n = 10) or sham FHD (n = 5) in early July 1983. Estrous cycles were monitored by measuring circulating progesterone concentrations from before FHD until September 1985. Group 2 ewes (n = 4) were subjected to FHD in October 1984. In late April 1985, blood samples were taken from all ewes at 1- to 4-h intervals from 1100 h to 0700 h of the following day to monitor diurnal changes of melatonin. Hypothalami were collected for histological evaluation of lesions. All Group 1 ewes (sham FHD and FHD) initiated normal estrous cycles in August and September 1983, and all ceased cycles by mid-February 1984. All sham FHD and 4 FHD ewes remained anestrus until August or September of 1984 and then resumed normal cycles. In contrast, 5 FHD ewes resumed cycles as early as April 1984 and then cycled intermittently or almost continuously. Two Group 2 ewes cycled continuously after FHD and 2 cycled infrequently. FHD ewes that showed prolonged breeding seasons had cuts that damaged the suprachiasmatic nucleus (SCN) and adjacent structures. Mean nocturnal (2000 h-0500 h) melatonin concentrations did not differ (p greater than 0.05) between sham FHD, FHD "normal season," and FHD "continuous cycle" ewes. In summary, damage to the SCN region by FHD during anestrus had no detectable effect on either onset or cessation of the next breeding season but greatly prolonged subsequent breeding seasons. Thus, the environmental signals that both initiated and terminated the 1983 breeding season apparently had been given before FHD was performed in midsummer. Damage to the SCN region during the breeding season caused some ewes to cycle continuously. The effects of FHD apparently were not due to disruption of melatonin secretion. FHD ewes that showed prolonged breeding seasons had normal seasonal changes of plasma prolactin concentrations. This suggests that different neural structures control seasonal patterns of gonadotropin and prolactin secretion.     

Effects of recent sexual experience and melatonin treatment of rams on plasma testosterone concentration, sexual behaviour and ability to induce ovulation in seasonally anoestrous ewes

The aim of this study was to determine whether advancing the seasonal changes associated with rams by treatment with exogenous melatonin and allowing the rams previous sexual experience would increase the proportion of anoestrous ewes ovulating in early July. North Country Mule ewes (n = 225) were grouped by live body weight and body condition score and allocated randomly to the following treatments: (i) isolated from rams (control; n = 25); (ii) introduced to rams (treatment 2); (iii) introduced to rams that had mated with ewes during the previous 2 days (treatment 3); (iv) introduced to rams implanted with melatonin (treatment 4); and (v) introduced to rams that were implanted with melatonin and had mated with ewes during the previous 2 days (treatment 5). Treatments 2-5 were replicated (2 x 25 ewes) and two rams were introduced to each replicate group. Introductions began on 4 July and were completed by 11 July. The rams were withdrawn from the ewes after 8 days. Melatonin was administered as a subcutaneous implant (Regulin((R))) on 22 May and again on 20 June. Blood samples were taken from all rams to determine plasma melatonin and testosterone concentrations (19 samples in 6 h). The behaviour of the sheep was videotaped continuously during the first 3 h after the ram was introduced. Ovulation was detected by an increase in plasma progesterone concentrations from < 0.5 ng ml(-1) to > 0.5 ng ml(-1). Mean +/- SE plasma melatonin concentrations were 649.7 +/- 281.4 and 18.3 +/- 2.4 pg ml(-1) in rams with and without melatonin implants, respectively (P < 0.001). Melatonin implants also increased plasma testosterone concentrations from 4.30 +/- 1.88 to 10.10 +/- 1.10 ng ml(-1) (P < 0.01), the libido of the rams and the proportion of ewes that ovulated in response to the rams (43 and 56% (treatments 4 and 5) versus 24% (treatments 2 and 3)). In conclusion, implanting rams with melatonin before introducing them to seasonally anoestrous ewes increases the proportion of ewes that ovulate in response to introduction of a ram, but previous sexual experience of rams appears to have little or no effect.     

Injections of alpha-melanocyte stimulating hormone affect pineal serotonin, melatonin and N-acetyltransferase activity

To determine if exogenously administered alpha-melanocyte stimulating hormone (alpha-MSH) affects nighttime pineal N-acetyltransferase activity, pineal levels of 5-hydroxytryptophan, serotonin and melatonin, and plasma prolactin levels, adult male hamsters were injected at 1900 hr (lights out 2000-0600 hr) with two doses of the peptide and killed at 0300 hr. The low dose of alpha-MSH (200 ng) produced a significant fall in pineal serotonin, pineal NAT activity and plasma prolactin values. The high dose of the peptide (20 micrograms) increased circulating prolactin titers and pineal serotonin levels and caused a concomitant decrease in pineal melatonin levels.     

The effect of melatonin implants on the response to the male effect and on the subsequent cyclicity of Rasa Aragonesa ewes implanted in April

Rasa Aragonesa ewes were used to evalutate whether treatment with melatonin implants in spring could modify: (i) the response to the male effect in terms of oestrous behaviour and ovulation rate; and (ii) the maintenance of sexual activity and ovulation rate at medium term, i.e. over the next 306 days. On 12 April, 42 ewes were divided into two groups, with (M; n = 21) or without (C; n = 21) a subcutaneous implant containing 18 mg melatonin. On 17 May (day 0), three aproned rams were introduced to each group to induce a ram effect. Ewes were observed for oestrus daily. The rams were removed 40 days later after which one aproned ram was introduced daily. Oestrous detection continued until 28 February, 306 days after the first male-female contact. The ovulation rate was determined by endoscopy in the first three cycles after ram introduction and in September-October and January. Progesterone was assayed from blood samples taken on 6 May, 10 and from day 0 to day 22 after ram introduction. Luteal activity before ram introduction was seen in 33% (M) and 29 (C)% of the ewes, respectively. Significantly more M ewes showed oestrous behaviour during the first 40 days after ram introduction (M: 100%; C: 62%; P < 0.01). Similar differences were observed for ewes anovulatory at ram introduction (M: 100%, C: 47%; P < 0.01). These differences were maintained over the three oestrous cycles in both groups. Treatment with melatonin implants was without detrimental effect on cyclic functions in the following breeding season, after seasonal anoestrus. Melatonin treatment significantly increased (P < 0.05) the mean ovulation rate of the first (1.62 +/- 0.11 versus 1.31 +/- 0.13), second (1.78 +/- 0.15 versus 1.36 +/- 0.15) and third cycles (M: 1.73 +/- 0.12 versus C: 1.27 +/- 0.14). There was a significant interaction between the effects of cyclicity at day 0 and melatonin treatment on the ovulation rate in the first cycle (P < 0.05). The mean ovulation rates of both groups were similar at the beginning (September) and middle (January) of the subsequent breeding season. Overall, the results confirmed that melatonin implants, combined with the ram effect, improved the reproductive parameters of reduced-seasonality ewes during spring mating, without impairing sexual activity or ovulation rate during the subsequent breeding season.     

Effect of neonatal melatonin administration on sexual development in the rat

In order to study the mechanisms by which melatonin modulates sexual development, 5-day-old female Wistar rats have been treated with a single s.c. injection of melatonin, 3 h before the darkness onset. Criteria for sexual development were the age of vaginal opening and the circulating levels of prolactin, LH, FSH and estradiol. Also, pineal melatonin content was measured. There was a precocious puberty (P less than 0.01) in melatonin-treated rats measured by the age of the vaginal opening. An increase in the number of estrous smears over the whole period studied was observed in melatonin-treated animals as compared to controls. Along with these modifications, there was decrease in pineal melatonin content and serum prolactin levels, on day 21 of life (P less than 0.05), with an increase in both parameters on day 30 of age, in melatonin-treated rats as compared to controls, with no modifications at any other time studied. No differences were detected for serum LH levels considering the whole period studied for both groups. There was a faster decrease in plasma FSH levels with age in melatonin-treated animals than in controls. Serum estradiol levels were decreased in the peripubertal period in melatonin-treated rats as compared to controls. All these data suggest that the modifications induced by neonatal melatonin administration on prolactin, FSH and estradiol could be responsible for the precocious puberty shown in this study.     

Length of melatonin exposure and onset of ovarian activity in anoestrous ewes

Adult anoestrous ewes (N = 30) were given intravaginal Silastic implants containing melatonin or empty implants (N = 7) during mid-anoestrus (4 July). Implants were removed 16 days later (Group 1), 36 days later (Group 2) or 93 days later (Group 3). Blood samples were taken twice weekly for progesterone assay to monitor onset of ovarian activity. The percentage of ewes in each group showing early onset of ovarian cyclicity was significantly correlated with length of exposure to melatonin.     

Postnatal development of female sheep pineal gland under natural inhibitory photoperiods: an immunocytochemical and physiological (melatonin concentration) study

The purpose of this study was to determine structural and immunocytochemical changes taking place during the day and at night in developing sheep pineal gland under natural non-stimulatory photoperiods (summer solstice). Additionally, the diurnal cycle of plasma melatonin levels was charted and differences between diurnal and nocturnal pineal melatonin concentrations were analyzed. 36 ewes of three different ages were examined: infants (1-6 months old), pubertal and early fertile age (9-24 months old) and adults (36-60 months old). Plasma and pineal gland melatonin levels were higher in pubertal sheep than in infants or adults. Pubertal sheep pineal glands were also heavier, contained a larger number of pinealocytes and interstitial cells and displayed more evident innervation and vascularisation than infants or adults. There was no difference in the number of pinealocytes and interstitial cells between animals killed during daylight or at night. Gland weight, pinealocyte nuclear profile areas and plasma melatonin concentrations were all significantly higher at night than during the day.     

Effect of constant-release implants of melatonin on seasonal cycles in reproduction, prolactin secretion and moulting in rams

Seasonal cycles in the size of the testes, blood plasma concentration of testosterone, FSH and prolactin, intensity of the sexual skin flush, timing of rutting behaviour and moulting of the body coat were recorded in Soay rams after s.c. implantation of melatonin contained in a Silastic envelope which increased the circulating blood levels of melatonin to 200-600 pg/ml for many months. Two groups of 8 adult rams were held under alternating periods of short days (8L:16D) and long days (16L:8D) to drive the seasonal cycles and the treatments with melatonin were initiated during the long or short days, and one group of 8 ram lambs was kept out of doors and given implants during the long days of summer (4 melatonin-implanted and 4 control (empty implants) rams per group). The treatments demonstrated that melatonin implants during exposure to long days resulted in a rapid 'switch on' of reproductive redevelopment similar to that produced by exposure to short days melatonin implants prevented the rams from showing the normal responses to changes in the prevailing photoperiod rendering them nonphotoperiodic; and long-term cyclic changes in testicular activity, prolactin secretion and other characteristics occurred in the melatonin-implanted rams; the pattern was similar to that previously observed in rams exposed to prolonged periods of short days. The overall results are consistent with the view that melatonin is the physiological hormone that relays the effects of changing photoperiod on reproduction and other seasonal features, and that continuous exogenous melatonin from an implant interferes with the normal 'signal' and produces an over-riding short-day response.     

Effect of maternal pinealectomy and reverse photoperiod on the circadian melatonin rhythm in the sheep and fetus during the last trimester of pregnancy

The present study tested the hypothesis that the nocturnal melatonin rhythm in the fetal sheep results from transfer across the placenta of melatonin from maternal circulation. Pregnant ewes were exposed to an artificial reverse photoperiod at about 100 days gestation (n = 6; lights on 10 h, 2200-0800 h PST). This treatment tested for entrainment in the ewe and its fetus of the 24-h pattern of melatonin production from the pineal gland. Other ewes were pinealectomized at 55 days post-breeding (n = 6), and similarly treated. Catheters were implanted and blood samples were collected between 117 and 142 days gestation at two 48-h periods, about every 0.5-4 h, to assess the pattern of melatonin in maternal and fetal circulations. In pineal-intact ewes and their fetuses, melatonin rhythms conformed to the reverse photoperiod, i.e. plasma melatonin concentrations were relatively low during the light period and significantly increased for the duration of darkness. In contrast, maternal pinealectomy abolished the melatonin rhythms in both the ewe and fetus; melatonin concentrations remained at or below the limits of detection. Pineal-intact sheep gave birth about 139 +/- 2 days (mean +/- SE, n = 4) at 1915 +/- 0.7 h and pinealectomized ewes (n = 5 of 6) lambed at 149 +/- 2 days at 0424 +/- 0.5 h. Finally, in lambs (n = 3) born to pinealectomized ewes, typical melatonin rhythms were present within the first week of life. The findings indicate that the maternal pineal gland is responsible for the 24-h pattern of melatonin in the ewe and its fetus during the last trimester of pregnancy.     

Melatonin and photorefractoriness: loss of response to the melatonin signal leads to seasonal reproductive transitions in the ewe

Experiments were conducted to examine whether the refractoriness of the Suffolk ewe to the reproductive effects of day length is associated with a deficit in the generation of the circadian rhythm of melatonin secretion or in the postpineal processing of this photoperiodic message. Using serum luteinizing hormone (LH) concentrations in ovariectomized ewes bearing constant-release estradiol implants as a marker of reproductive induction, ewes with intact pineal glands were found to become unresponsive to fixed artificial photoperiods that initially had been either inductive (short days) or inhibitory (long days). The loss of the photoperiodic response was not associated with notable changes in the 24-h secretory pattern of melatonin, which remained characteristically low throughout the day and rose at night. In pinealectomized ewes, nightly infusion of a stimulatory pattern of melatonin (simulating that seen on short days) initially provoked reproductive induction; this response then lessened over much the same time course that pineal intact ewes became refractory to short days. These results support the hypothesis that photorefractoriness reflects a deficit in the postpineal processing of the photoperiodic message. Further, in view of recent evidence that photorefractoriness normally leads to both onset and cessation of the breeding season in Suffolk ewes maintained outdoors, these findings suggest that the loss of response to the melatonin signal contributes to at least one of these reproductive transitions, the cessation of the breeding season, under natural environmental conditions.     

Nightly duration of pineal melatonin secretion determines the reproductive response to inhibitory day length in the ewe

The pineal controls the reproductive response of ewes to both stimulatory (short) and inhibitory (long) day lengths. Melatonin, a pineal hormone whose nocturnal secretion is entrained by photoperiod, mediates the effect of stimulatory photoperiod. We now report that melatonin also mediates the effect of inhibitory day length, monitored as response to estradiol negative feedback on luteinizing hormone (LH) secretion. Ovariectomized, estradiol-implanted ewes were pinealectomized and intravenously infused with melatonin to restore the nightly melatonin rise. Following transfer from short to long days, and a concurrent switch from short- to long-day melatonin patterns, LH dropped precipitously in pinealectomized ewes, matching the photoinhibitory response of pineal intact controls. LH dropped similarly in pinealectomized ewes when long-day melatonin was infused under short days. Pinealectomized ewes transferred from long to short days displayed a marked LH rise, provided melatonin was also switched to the short-day pattern. LH remained suppressed if long-day melatonin was infused following transfer to short days. These data indicate the nighttime melatonin rise mediates reproductive responses to inhibitory, as well as stimulatory photoperiods; they further suggest the duration of this rise controls suppression of LH under long days. Rather than being strictly pro- or antigonadal, the pineal participates in measuring day length.     

Effect of duration of melatonin treatment on the onset and duration of oestrous cyclicity in ewes.

Forty-two Scottish Blackface ewes that lambed outdoors in March were removed from their lambs at the end of April and housed under natural daylength at 57 degrees N. Treatments (n = 7 ewes per treatment) commenced on 1 May and comprised daily oral dosing at 15:00 h with 3 mg melatonin dissolved in water and ethanol (4:1, v/v) for 30, 60, 90, 120 or 150 days. Control ewes received the vehicle alone. Ovarian activity was assessed by laparoscopy at monthly intervals with an additional interim observation in mid-July. Blood was sampled three times a week by jugular venepuncture and assayed for progesterone, prolactin and follicle-stimulating hormone (FSH). Luteinizing hormone (LH) was determined in blood samples collected at 15 min intervals for 10 h on days 28, 60, 91, 119 and 150. Thirty days of melatonin treatment delayed (P < 0.01) first ovulation by about 1 month (mean interval +/- SEM from 1 May to progesterone > 1 ng ml-1, 165 +/- 4.5 days versus 132 +/- 9.2 days for controls). None of the ewes that received melatonin for 60 days ovulated before the end of melatonin treatment, but subsequently six of them did; the mean interval from 1 May to increased progesterone concentration was 75 +/- 1.2 days. All ewes receiving melatonin for 90, 120 and 150 days ovulated with corresponding mean intervals of 83 +/- 2.7, 85 +/- 1.3 and 87 +/- 2.2 days, respectively (P < 0.001 compared with controls).(ABSTRACT TRUNCATED AT 250 WORDS)     

How does melatonin code for day length in the ewe: duration of nocturnal melatonin release or coincidence of melatonin with a light-entrained sensitive period?

The main objective of the study was to test the hypothesis that the phase of melatonin release with respect to the light-dark cycle mediates the effects of photoperiod on the reproductive response of the ewe. To test the phase hypothesis, we eliminated endogenous melatonin secretion by pinealectomy and then restored physiological levels of serum melatonin with rises of the same duration but at different phases of the light-dark cycle (either at night or in the middle of the day). Serum melatonin patterns were determined by radioimmunoassay in samples taken hourly for 24 h. The reproductive state was monitored by measuring serum luteinizing hormone (LH) in ovariectomized ewes treated with constant-release estradiol implants. Infusion of a long-day pattern of melatonin was equally effective in maintaining reproductive suppression when given during the night or the middle of the day. LH remained low for approximately 150 days and then rose as ewes became refractory to the inhibitory melatonin signal. These results do not support the phase hypothesis. Rather, they are consistent with the hypothesis that the duration of the nocturnal secretion of melatonin codes for day length.     

Pituitary response to LHRH, LH pulsatility and plasma melatonin and prolactin changes in ewe lambs treated with melatonin implants to delay puberty

Prepubertal ewe lambs were treated with empty or filled melatonin implants. The implants were placed s.c. at birth and pituitary responsiveness to various doses of LHRH, LH/FSH pulsatility and prolactin and melatonin secretion were examined at 10, 19, 28, 36 and 45 weeks of age. Control animals (N = 10) showed no consistent alteration in pituitary responsiveness to LHRH during development. Ewes treated with melatonin (N = 10) had puberty onset delayed by 4 weeks (P less than 0.03) but no effect of melatonin on LH or FSH response to LHRH injection was observed at any stage of development. In the control and melatonin-treated ewe lambs the responses to LHRH injection were lower during darkness than during the day at all stages of development. No consistent differences in LH or FSH pulsatility were observed between treatment groups or during development. Prolactin concentrations, however, failed to decrease at the time of puberty (autumn) in the melatonin-treated group. Melatonin-treated ewe lambs maintained normal rhythmic melatonin production which was superimposed on a higher basal concentration and showed the same increase in melatonin output with age as the control ewes. These results indicate that the delayed puberty caused by melatonin implants is not due to decreased pituitary responsiveness to LHRH or to dramatic changes in basal LH or FSH secretion.