Alterations of estrous activity in the ewe by circadian-based manipulation of the endogenous pacemaker.

The timing of reproductive activity in the seasonal breeding Romney Marsh ewe depends on the measurement of photoperiodic time. In this experiment, artificial light and dark signals are provided in a measured sequence at an inappropriate time of year to induce breeding out of phase with environmental photoperiod. The endogenous circadian responses and reproductive effects are documented. One group (Group A, control) of 6 Romney Marsh ewes was held in natural photoperiod throughout the experiment. For 8 weeks centered about the winter solstice (Stage 1), an additional 18 animals (Groups B, C, and D) were exposed to an artificial earlier dawn. Measurements of endogenous melatonin performed under acutely extended darkness confirmed a phase advance of the endogenous circadian pacemaker of the suprachiasmatic nucleus compared to control animals. In Stage 2, to the summer solstice (21 December), Group B animals were returned to natural photoperiod, Group C animals were subjected to an earlier artificial dusk, and Group D animals were subjected to an artificial delayed dawn. Melatonin measurements during Stage 2 confirmed that onset and offset times for Group C were earlier and that onset and offset times for Group D were delayed compared to corresponding times for Group B animals. Ovarian activity was monitored throughout. During Stage 2, Groups C and D commenced reproductive activity in mid-spring, and this continued until the experimental conditions changed. Groups A and B commenced reproductive activity at the normal timing in the subsequent autumn. Although not exclusive, these results are consistent with a coincidence model to explain the timing of seasonal breeding in this species with a dusk-located phase of the endogenous pacemaker sensitive to both light and melatonin. The temporal relationship between circadian alterations and the environmental photoperiod warrants further investigation as an explanation for seasonal breeding.

The coincidence of light and melatonin with a specific phase of the circadian pacemaker is important for the timing of seasonal breeding in the ewe.

The timing of reproductive activity in seasonal breeding sheep relies on daily photoperiodic signals being relayed to provide information on the time of year. Although light and melatonin are involved, the exact mechanism is not understood. In this experiment, three groups of 6 Romney Marsh ewes, a highly seasonal breed, were provided with 8 weeks of short nights (9.6-9.8 h, by artificially advancing dawn) around the winter solstice, near the end of their natural breeding season. One group of animals was infused to a physiological level with melatonin for 5 h during the afternoon prior to the onset of dark, while a second group was identically infused but for 5 h from the time of lights on. A third group received the short-night treatment only. Following the short-night treatment, all groups were exposed to long nights (> 14 h, by delaying dawn) until the summer solstice. Ovarian activity, assessed by progesterone monitoring twice weekly, showed that the noninfused and the morning-infused groups displayed renewed reproductive activity in response to the short-night/long-night treatment. There was no renewed ovarian activity in the afternoon-infused group, indicating that the time of day that melatonin is present, rather than the duration of melatonin exposure, is an important signal in the control of reproductive timing. Measurements of a marker of the endogenous circadian pacemaker, by melatonin measurements under acutely extended darkness, revealed that the short-night treatments phase advanced the onset of the pacemaker in all groups such that the afternoon phase of the pacemaker was coincident with light. The results provide strong support for the model that proposes that an afternoon-located sensitive phase of the pacemaker is responsible for the relay of photoperiodic signals in the timing control of seasonal breeding. The model proposes that the reproductive axis be primed during short nights when the sensitive phase is coincident with light in the afternoon so ovarian activity can be induced when the sensitive phase is located within the longer nights of autumn and coincident with endogenous melatonin.

Is a critical interval of the circadian pacemaker at dusk responsive to light and melatonin responsible for the timing of estrus in the Romney Marsh ewe?

Two experiments, using Romney Marsh ewes, tested for the existence and role of a critical interval of the circadian pacemaker located near dusk that may be integrally involved in the precise timing of the breeding season. Groups of Romney Marsh ewes (n = 6) were provided with exogenous melatonin by injection at dusk (Experiment 1) or by infusion at dawn or subjected to extended darkness at dawn (Experiment 2) from the winter to the summer solstice before being exposed to natural photoperiod at latitude 35 degrees S. Other than the experimental protocols, all animals were held in natural photoperiod. The onset of the breeding season (defined as cyclic ovarian activity as indicated by plasma progesterone monitoring) was normal in those animals treated with morning melatonin but was delayed in those animals treated with melatonin at dusk or extended darkness at dawn compared to controls in natural photoperiod (p < .01). Exogenous melatonin at dusk was associated with a phase advance of the onset of the circadian pacemaker (as measured by endogenous melatonin in acutely extended darkness); additional darkness at dawn was associated with a phase delay of both the onset and the offset of the circadian pacemaker. Exogenous morning melatonin did not change the phase of the circadian pacemaker relative to the controls. The results are consistent with an external coincidence model of seasonal breeding in which a critical interval of the circadian pacemaker requires exposure to light during spring/summer to time estrus correctly. The proposed critical interval appears to be located near dusk in this model and is phase locked to the circadian pacemaker. The effect of the exogenous melatonin on the timing of the breeding season is similar to darkness when administered at dusk but is not equivalent to darkness at dawn. The timing of anestrus was not affected by any of the experimental treatments and may reflect a common response to an environmental influence.

Collection and analysis of semen from the common marmoset (Callithrix jacchus).

The authors examined the seminal characteristics of 16 male common marmosets (Callithrix jacchus) to provide baseline data for future studies of the reproductive biology of this species. Semen samples were collected by electroejaculation. There was considerable inter- and intra-male variation in all seminal characteristics. The median seminal volume was 30 microliters (range 8 to 85 microliters), and the median total sperm count was 5.1 x 10(6) sperm (range, 0.1 to 43 x 10(6]. The median progressive sperm motility was 48% (range, 10% to 76%), and 49% of the sperm exhibited normal morphology (range, 24% to 81%). Three types of sperm head abnormalities and eight types of tail defects were noted. Tail defects were common (median, 50%; range, 17% to 76%), whereas head defects were relatively rare (median, 4.5%; range, 0% to 24%). The results indicate that semen samples can be routinely collected from this species, but considerable inter- and intra-male variation can be expected. It is therefore important to examine several semen samples from each male marmoset to obtain an accurate seminal picture.

Environmental temperature has an influence on timing of the first ovulation of seasonal estrus in the mare.

A 10-yr retrospective analysis of the time of onset of seasonal ovulation derived from the records of a Thoroughbred horse stud at latitude 35 degrees S revealed a significant variation between years. The onset of seasonal ovulation was closely related to both the maximum (correlation coefficient r=0.56; P=0.09) and minimum (r=-0.67; P<0.01) environmental temperatures. The trend of the temperature rise and the levels attained in the weeks immediately prior to ovulation were similar for both the environmental maximum and minimum temperatures in all years of the study. Environmental temperature may therefore play an important adjuvant zeitgeber for the timing of the first ovulation of estrus in the mare.

Plasma melatonin exhibits a diurnal secretion in the common marmoset (Callithrix jacchus): relationship to the rest-activity cycle.

The secretion of plasma melatonin exhibits a diurnal variation in the common marmoset (Callithrix jacchus jacchus) (n = 12) with mean values of 125.8 fmol/ml during darkness and below assay sensitivity (mean 70.5 fmol/ml) during the light period. Both melatonin secretion and the rest activity cycle appeared to be phase advanced with relation to the light dark cycle which is consistent with an endogenous circadian period (tau) of less than 24 h in this species.

Effect of exogenous melatonin and extending the dark period at dusk before the summer solstice on the onset of oestrus in Romney Marsh ewes.

Groups of Romney Marsh ewes (n = 24) were injected with exogenous melatonin or subjected to extended darkness or a combination of both at dusk from the winter to the summer solstice before being re-exposed to the natural photoperiod at 35 degrees S. The animals were at all times exposed to natural dawn. The onset of subsequent oestrus was delayed when compared with controls subjected to the natural photoperiod. Either melatonin or darkness or a combination were equally effective at delaying oestrus. The results are consistent with a hypothesis based on an external coincidence model of seasonal breeding involving the coincidence of a light-sensitive phase set close to dusk that, when exposed to light in spring and summer, prepares the reproductive axis to respond correctly to the direct effects of the inductive photoperoid in autumn. The offset of the time of oestrus was not affected by the experimental treatments nor was the timing of the subsequent reproductive season. Since melatonin was as effective as darkness at influencing the assumed photosensitive signal, even in the presence of natural light, melatonin may act through the light reception system.

Human plasma melatonin and urinary 6-sulphatoxy melatonin: studies in natural annual photoperiod and in extended darkness.

OBJECTIVES: The aims of the study were (1) to examine the human plasma melatonin rhythm at the equinoxes and the solstices in the natural photoperiod (at 35 degrees S); (2) to examine melatonin rhythms in the same subjects under extended darkness conditions to expose any suppressive (gating) effects of light at any time of the year; (3) to undertake a rigorous examination of the relationship between plasma melatonin and the urinary metabolite 6-sulphatoxy melatonin at varying times of the year. DESIGN: At the equinoxes and solstices, unrestricted subjects had hourly urine collections followed by venous blood sampling taken under natural light conditions for 24 hours. Following a 24 hour interval, a similar collection regime was performed with subjects held under conditions of extended darkness (5 hours darkness prior to natural sunset and following natural sunrise) for a further 24 hours. SUBJECTS: Groups of four (minimum) to six female volunteers (age range 18-35 years) were studied, who had a normal lifestyle, no history of depression, and were not taking any medication or recently engaged in shiftwork. MEASUREMENTS: The plasma was assayed for melatonin and the urine samples for 6-sulphatoxy melatonin by radioimmunoassay. RESULTS: The onset of natural melatonin secretion was delayed until after sunset at all seasons but was earlier in summer, and not different from the time of sunset in extended darkness. The offset of melatonin secretion under natural conditions occurred at sunrise in autumn and winter but was delayed until after sunrise during spring and summer, particularly in extended darkness. No significant changes in the duration of melatonin secretion were observed between seasons nor between the duration of melatonin secretion under natural photoperiod or extended darkness. The measurement of 6-sulphatoxy melatonin proved to be a close indicator of the phase and amplitude of secretion of plasma melatonin. Both onset and offset times of 6-sulphatoxy melatonin were delayed compared to the times when plasma melatonin was detectable/undetectable. A good correlation exists between the total plasma melatonin secretion and that of 6-sulphatoxy melatonin. CONCLUSIONS: The results suggest evidence for a suppressive (gating) effect of light at dawn only during summer which was associated with a phase advance of the onset of melatonin secretion at this time of year. The lack of a major gating effect of environment light on melatonin secretion, and the unchanging duration of secretion through the year in the normally entrained human, highlight differences between the human and those photoperiodic animal species which breed seasonally. Urinary 6-sulphatoxy melatonin proved to be a good indicator of plasma melatonin levels under rigorous examination and is confirmed as a useful clinical measure.

Plasma melatonin profiles of Romney Marsh sheep in natural photoperiod and in acutely extended darkness.

Plasma melatonin was measured at the summer and winter solstices and the autumn and spring equinoxes in Romney Marsh sheep held under natural conditions in South Australia (35 degrees S). The amount of melatonin detected was generally related to the extent of natural darkness, though the melatonin onset was particularly delayed after dusk in winter compared with other seasons. The duration of detectable melatonin was shorter in summer than at any other season. After each initial 24 h sampling, the sheep were resampled for a further 24 h in acutely extended darkness to mark the phase and duration of suprachiasmatic nuclei activity which is believed to be the source of the melatonin signal. The onset of high plasma melatonin was earlier than the time of natural sunset in spring and summer, but not different from the time of natural sunset in autumn and winter. The offset of high plasma melatonin was later than the time of natural sunrise at all times of year and particularly so in summer. Under the extended dark conditions, the duration of detectable melatonin was longer than that under natural photoperiod at all seasons of the year and the duration of melatonin was again shorter in summer than winter. If melatonin measurements under the conditions of extended darkness do reflect the phase and duration of suprachiasmatic nuclei function then the natural light of the photoperiod can, particularly during long photoperiod conditions, mask the expression of the pacemaker. The findings may have implications for the timing of the breeding season in Romney Marsh sheep.

Melatonin and photoperiodic time measurement: seasonal breeding in the sheep.

Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status--the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.

Plasma melatonin in the horse: measurements in natural photoperiod and in acutely extended darkness throughout the year.

Plasma melatonin was measured at the winter and summer solstices and the autumn and spring equinoxes in four mares held under natural conditions at 35 degrees S. At all seasons the onset of the nightly elevated melatonin was coincident with or after the time of sunset and the melatonin offset after the time of sunrise. The duration of elevated melatonin was not different from the duration of natural scotophase for each season, with the duration of elevated melatonin longer in winter than the other seasons. Immediately following each 24 hr sampling two mares were resampled in acutely extended darkness to determine the melatonin profile of the endogenous rhythm of the circadian pacemaker, originating from the suprachiasmatic nuclei (SCN). At each season melatonin secretion commenced earlier and decreased later than that measured under the natural photoperiodic condition, suggesting that the expression of the melatonin rhythm is normally gated by natural environmental light both at dusk and dawn. The interval from the onset of melatonin measured under acutely extended darkness to the time of sunset was greater in the spring/summer than the autumn/winter suggesting a possible alternating signal throughout the year. Thus the mare appears to exhibit a similar interaction between endogenous circadian rhythmic activity and the natural photoperiod as the ewe which may underlie the mechanism for timing reproductive activity through the year.