Features of the daily rhythms of blood melatonin and glucose in goats during different natural photoperiod

The scope of the present study was to explore the features of the daily rhythms of plasma melatonin and blood glucose in goats during different natural photoperiod imposed by the four seasons. Five Sarda breed pluriparus female goats, 2 years old, with a mean body weight 40.5 ± 2.3 kg, lead to a natural pasture, were cannulated the day before the start of the study for sample collection performed during spring and autumn equinoxes, and winter and summer solstices for a 48-h period. On blood samples, melatonin and glucose concentrations were assessed. Our results indicate a daily rhythm of both parameters studied which characteristics were influenced by the different photoperiod due to season. During the experimental period, the two parameters showed a negative correlation, despite the onset of melatonin concentration was linked to the sunset, whereas the onset and offset of glucose were not linked to the photoperiod. In conclusion, we can claim that the two parameters are driven by different entrainable systems, but further studies are necessary to establish their correlation.     

Different behavior of body temperature and total locomotor activity daily rhythms during light/dark cycle in stabled Oryctolagus cuniculus

In order to investigate the potential causal link between the rhythm of activity and body temperature, we simultaneously recorded rectal temperature and total locomotor activity in five clinically healthy female rabbits (blue Vienna breed), 12 week old and mean body weight 2.7 ± 0.3. Animals were housed in individual cages (90?×?50?×?35 cm) under natural 12/12 light/dark cycle. Total locomotor activity was monitored for 15 days by an activity data logger. On day 1, 5, 10, and 15 rectal temperature was recorded every 2 h for a 24-h period. Application of single cosinor method showed a nocturnal daily rhythm of rectal temperature with a range of oscillation of about 1 °C, acrophase after dusk and low robustness value. The daily rhythm of locomotor activity showed its acrophase in the middle of the scotophase and a high robustness value. This information improves the knowledge available on the circadian biology of rabbits useful in the evaluation of physiology of this species.     

The Circadian Body Temperature Rhythm in the Elderly: Effect of Single Daily Melatonin Dosing

The present study is part of a more extensive investigation dedicated to the study and treatment of age‐dependent changes/disturbances in the circadian system in humans. It was performed in the Tyumen Elderly Veteran House and included 97 subjects of both genders, ranging from 63 to 91 yrs of age. They lived a self‐chosen sleep‐wake regimen to suit their personal convenience. The experiment lasted 3 wks. After 1 control week, part of the group (n=63) received 1.5 mg melatonin (Melaxen?) daily at 22:30 h for 2 wks. The other 34 subjects were given placebo. Axillary temperature was measured using calibrated mercury thermometers at 03:00, 08:00, 11:00, 14:00, 17:00, and 23:00 h each of the first and third week. Specially trained personnel took the measurements, avoiding disturbing the sleep of the subjects. To evaluate age‐dependent changes, data obtained under similar conditions on 58 young adults (both genders, 17 to 39 yrs of age) were used. Rhythm characteristics were estimated by means of cosinor analyses, and intra‐ and inter‐individual variability by analysis of variance (ANOVA). In both age groups, the body temperature underwent daily changes. The MESOR (36.38±0.19°C vs. 36.17±0.21°C) and circadian amplitude (0.33±0.01°C vs. 0.26±0.01°C) were slightly decreased in the elderly compared to the young adult subjects (p<0.001). The mean circadian acrophase was similar in both age groups (17.19±1.66 vs. 16.93±3.08 h). However, the inter‐individual differences were higher in the older group, with individual values varying between 10:00 and 23:00 h. It was mainly this phase variability that caused a decrease in the inter‐daily rhythm stability and lower group amplitude. With melatonin treatment, the MESOR was lower by 0.1°C and the amplitude increased to 0.34±0.01°C, a similar value to that found in young adults. This was probably due to the increase of the inter‐daily rhythm stability. The mean acrophase did not change (16.93 vs. 16.75 h), although the inter‐individual variability decreased considerably. The corresponding standard deviations (SD) of the group acrophases were 3.08 and 1.51 h (p<0.01). A highly significant correlation between the acrophase before treatment and the phase change under melatonin treatment indicates that this is due to a synchronizing effect of melatonin. Apart from the difference in MESOR, the body temperature rhythm in the elderly subjects undergoing melatonin treatment was not significantly different from that of young adults. The data clearly show that age‐dependent changes mainly concern rhythm stability and synchronization with the 24 h day. A single daily melatonin dose stabilizes/synchronizes the body temperature rhythm, most probably via hypothermic and sleep‐improving effects.     

Analysis of trough and peak of plasma melatonin circadian rhythm in ewes

This study evaluates the pattern of plasma melatonin during the trough and the peak of its daily rhythm. Blood samples from 8 ewes were collected every 3 h for a 48-h period. On the third day, blood samples were collected from 10:00 to 13:00 (trough) and from 20:00 to 23:00 (peak) every 20 min. Our results showed a robust daily rhythm of melatonin in both days of monitoring, with nocturnal acrophase. During the trough, a significant decrease was observed starting from the 10:40 with a progressive decrease about every 40 min. During the peak of the plasma melatonin daily rhythm, an increase was observed starting from the 20:40 with a progressive increase about every 40 min. These data could be taken in consideration to monitor the plasma melatonin variations during the 24 h, and for the administration of melatonin for breeding in ewes.     

Circadian rhythms of melatonin in haemolymph and optic lobes of Chinese mitten crab (Eriocheir sinensis) and Chinese grass shrimp (Palaemonetes sinensis)

This study is the first to examine the circadian rhythms of melatonin in Eriocheir sinensis and Palaemonetes sinensis, two economically important crustaceans. We collected haemolymph and optic lobes from both species every 4 h over a whole day cycle. Melatonin content was measured with high-performance liquid chromatography. E. sinensis haemolymph exhibited significant (p < 0.05) peaks in melatonin at 16:00 (0.180 ± 0.020 μg·mL^?1) and 24:00 (0.244 ± 0.055 μg·mL^?1), while eyestalks had significant peaks at 16:00 (72.377 ± 18.100 μg·eyestalk^?1) and 24:00 (98.756 ± 30.271 μg·eyestalk^?1). In P. sinensis, melatonin peaked significantly only at 16:00 in optic lobes (12.493 ± 1.475 μg·eyestalk^?1) (p < 0.05); no significant peaks were present in haemolymph. Thus, both E. sinensis and P. sinensis exhibit species-specific melatonin rhythms. Time of day should therefore be considered when examining the physiological status of both crustaceans, given the potential influence of fluctuating daily melatonin concentrations.     

Relationship between MTNR1A melatonin receptor gene polymorphism and seasonal reproduction in different goat breeds

The reproductive activity of goats bred in temperate latitude follows a seasonal pattern, influenced by annual variation in day length. Daily variation in pineal melatonin secretion is the neuroendocrine signal recognized by animals through the link between this hormone and melatonin receptor 1a (MTNR1A). A total of 345 goats of different breeds (225 Sarda, 30 Saanen, 30 Chamois Coloured, 30 Maltese and 30 Nubian) with a kidding period in October-December or January-March were analysed to verify if a link exists between the structure of the receptor gene and reproductive activity. The main part of exon II of MTNR1A gene was amplified by PCR and then digested with MnlI and RsaI to prove the presence of restriction sites. Sequencing of 20 cloned samples and 20 purified samples permitted comparison with previously published sequences. No polymorphism was found using MnlI enzyme, as all 345 samples showed the cleavage site in position 605 and all the goats were MM genotype. However, using RsaI enzyme, some Sarda goats, showed a polymorphic site in position 53. Nine Sarda goats were R/r genotype, lacking this cleavage site only in one allele, while the other animals, both Sarda and the other breeds, presented the cleavage site in both the alleles and were thus R/R genotype. No r/r genotype was found in any of the breeds. In Sarda goats the allelic frequency was 0.98 for R allele and 0.02 for r allele; genotypic frequency was 96.00% for R/R genotype and 4.00% for R/r genotype. A strong link emerged from statistical analysis (P<0.001) between R/r genotype and reproductive activity, which was strongly influenced by photoperiod. Sequencing indicated six nucleotide changes that did not induce any amino acid change. Data showed that polymorphism was present and that it influences reproductive activity only in the Sarda breed.     

Change of serum mitochondrial uncoupling protein 1 (UCP1) levels and daily rhythm of rectal and cutaneous temperatures in Equus caballus and Capra hyrcus

The aim of the present study was to monitor the daily rhythm of rectal and cutaneous temperatures together with the changes in the serum levels of mitochondrial uncoupling protein 1 (UCP1) in equine and goat species. On five gelding horses and five female goat rectal and cutaneous temperatures were recorded at 4 h intervals for a 48 h period. At the same time points blood samples were collected from each animal. Daily rhythms of rectal and cutaneous temperatures were observed in both species, in both day of monitoring. Mesor value of rectal temperature was statistically higher of about 4 °C than cutaneous temperature, and acrophase was postponed of about 2 h in both day of monitoring and in both species. UCP1 did not show daily rhythmicity in horses and goats. We can speculate that the thermogenesis due to thermal system is auxiliary to keeping the body temperature daily rhythm.     

Effects of obstructive sleep apnea on endogenous circadian rhythms assessed during relaxed wakefulness; an exploratory analysis

ABSTRACT

Obstructive sleep apnea (OSA) is associated with hypertension, cardiovascular disease, and a change in the 24 h pattern of adverse cardiovascular events and mortality. Adverse cardiovascular events occur more frequently in the middle of the night in people with OSA, earlier than the morning prevalence of these events in the general population. It is unknown if these changes are associated with a change in the underlying circadian rhythms, independent of behaviors such as sleep, physical activity, and meal intake. In this exploratory analysis, we studied the endogenous circadian rhythms of blood pressure, heart rate, melatonin and cortisol in 11 participants (48 ± 4 years; seven with OSA) throughout a 5 day study that was originally designed to examine circadian characteristics of obstructive apnea events. After a baseline night, participants completed 10 recurring 5 h 20 min behavioral cycles divided evenly into standardized sleep and wake periods. Blood pressure and heart rate were recorded in a relaxed semirecumbent posture 15 minutes after each scheduled wake time. Salivary melatonin and cortisol concentrations were measured at 1–1.5 h intervals during wakefulness. Mixed-model cosinor analyses were performed to determine the rhythmicity of all variables with respect to external time and separately to circadian phases (aligned to the dim light melatonin onset, DLMO). The circadian rhythm of blood pressure peaked much later in OSA compared to control participants (group × circadian phase, p < .05); there was also a trend toward a slightly delayed cortisol rhythm in the OSA group. Rhythms of heart rate and melatonin did not differ between the groups. In this exploratory analysis, OSA appears to be associated with a phase change (relative to DLMO) in the endogenous circadian rhythm of blood pressure during relaxed wakefulness, independent of common daily behaviors.     

The circadian body temperature rhythm in the elderly: effect of single daily melatonin dosing

The present study is part of a more extensive investigation dedicated to the study and treatment of age-dependent changes/disturbances in the circadian system in humans. It was performed in the Tyumen Elderly Veteran House and included 97 subjects of both genders, ranging from 63 to 91 yrs of age. They lived a self-chosen sleep-wake regimen to suit their personal convenience. The experiment lasted 3 wks. After 1 control week, part of the group (n=63) received 1.5 mg melatonin (Melaxen) daily at 22:30 h for 2 wks. The other 34 subjects were given placebo. Axillary temperature was measured using calibrated mercury thermometers at 03:00, 08:00, 11:00, 14:00, 17:00, and 23:00 h each of the first and third week. Specially trained personnel took the measurements, avoiding disturbing the sleep of the subjects. To evaluate age-dependent changes, data obtained under similar conditions on 58 young adults (both genders, 17 to 39 yrs of age) were used. Rhythm characteristics were estimated by means of cosinor analyses, and intra- and inter-individual variability by analysis of variance (ANOVA). In both age groups, the body temperature underwent daily changes. The MESOR (36.38+/-0.19 degrees C vs. 36.17+/-0.21 degrees C) and circadian amplitude (0.33+/-0.01 degrees C vs. 0.26+/-0.01 degrees C) were slightly decreased in the elderly compared to the young adult subjects (p<0.001). The mean circadian acrophase was similar in both age groups (17.19+/-1.66 vs. 16.93+/-3.08 h). However, the inter-individual differences were higher in the older group, with individual values varying between 10:00 and 23:00 h. It was mainly this phase variability that caused a decrease in the inter-daily rhythm stability and lower group amplitude. With melatonin treatment, the MESOR was lower by 0.1 degrees C and the amplitude increased to 0.34+/-0.01 degrees C, a similar value to that found in young adults. This was probably due to the increase of the inter-daily rhythm stability. The mean acrophase did not change (16.93 vs. 16.75 h), although the inter-individual variability decreased considerably. The corresponding standard deviations (SD) of the group acrophases were 3.08 and 1.51 h (p<0.01). A highly significant correlation between the acrophase before treatment and the phase change under melatonin treatment indicates that this is due to a synchronizing effect of melatonin. Apart from the difference in MESOR, the body temperature rhythm in the elderly subjects undergoing melatonin treatment was not significantly different from that of young adults. The data clearly show that age-dependent changes mainly concern rhythm stability and synchronization with the 24 h day. A single daily melatonin dose stabilizes/synchronizes the body temperature rhythm, most probably via hypothermic and sleep-improving effects.     

Older Poor-Sleeping Women Display a Smaller Evening Increase in Melatonin Secretion and Lower Values of Melatonin and Core Body Temperature Than Good Sleepers

Melatonin concentration and core body temperature (CBT) follow endogenous circadian biological rhythms. In the evening, melatonin level increases and CBT decreases. These changes are involved in the regulation of the sleep-wake cycle. Therefore, the authors hypothesized that age-related changes in these rhythms affect sleep quality in older people. In a cross-sectional study design, 11 older poor-sleeping women (aged 62–72 yrs) and 9 older good-sleeping women (60–82 yrs) were compared with 10 younger good-sleeping women (23–28 yrs). The older groups were matched by age and body mass index. Sleep quality was assessed by the Pittsburgh Sleep Quality Index questionnaire. As an indicator of CBT, oral temperature was measured at 1-h intervals from 17:00 to 24:00?h. At the same time points, saliva samples were collected for determining melatonin levels by enzyme-linked immunosorbent assay (ELISA). The dim light melatonin onset (DLMO), characterizing the onset of melatonin production, was calculated. Evening changes in melatonin and CBT levels were tested by the Friedman test. Group comparisons were performed with independent samples tests. Predictors of sleep-onset latency (SOL) were assessed by regression analysis. Results show that the mean CBT decreased in the evening from 17:00 to 24:00?h in both young women (from 36.57°C to 36.25°C, p < .001) and older women (from 36.58°C to 35.88°C, p < .001), being lowest in the older poor sleepers (p < .05). During the same time period, mean melatonin levels increased in young women (from 16.2 to 54.1 pg/mL, p < .001) and older women (from 10.0 to 23.5 pg/mL, p < .001), being lowest among the older poor sleepers (from 20:00 to 24:00?h, p < .05 vs. young women). Older poor sleepers also showed a smaller increase in melatonin level from 17:00 to 24:00?h than older good sleepers (mean?±?SD: 7.0?±?9.63 pg/mL vs. 15.6?±?24.1 pg/mL, p = .013). Accordingly, the DLMO occurred at similar times in young (20:10?h) and older (19:57?h) good-sleeping women, but was delayed ～50?min in older poor-sleeping women (20:47?h). Older poor sleepers showed a shorter phase angle between DLMO and sleep onset, but a longer phase angle between CBT peak and sleep onset than young good sleepers, whereas older good sleepers had intermediate phase angles (insignificant). Regression analysis showed that the DLMO was a significant predictor of SOL in the older women (R²?=?0.64, p < .001), but not in the younger women. This indicates that melatonin production started later in those older women who needed more time to fall asleep. In conclusion, changes in melatonin level and CBT were intact in older poor sleepers in that evening melatonin increased and CBT decreased. However, poor sleepers showed a weaker evening increase in melatonin level, and their DLMO was delayed compared with good sleepers, suggesting that it is not primarily the absolute level of endogenous melatonin, but rather the timing of the circadian rhythm in evening melatonin secretion that might be related to disturbances in the sleep-wake cycle in older people. (Author correspondence: mdittmar@zoologie.uni-kiel.de)     

Dim Light Melatonin Onset in Alcohol-Dependent Men and Women Compared with Healthy Controls

Sleep disturbances in alcohol-dependent (AD) individuals may persist despite abstinence from alcohol and can influence the course of the disorder. Although the mechanisms of sleep disturbances of AD are not well understood and some evidence suggests dysregulation of circadian rhythms, dim light melatonin onset (DLMO) has not previously been assessed in AD versus healthy control (HC) individuals in a sample that varied by sex and race. The authors assessed 52 AD participants (mean?±?SD age: 36.0?±?11.0 yrs of age, 10 women) who were 3–12 wks since their last drink (abstinence: 57.9?±?19.3 d) and 19 age- and sex-matched HCs (34.4?±?10.6 yrs, 5 women). Following a 23:00–06:00?h at-home sleep schedule for at least 5 d and screening/baseline nights in the sleep laboratory, participants underwent a 3-h extension of wakefulness (02:00?h bedtime) during which salivary melatonin samples were collected every 30?min beginning at 19:30?h. The time of DLMO was the primary measure of circadian physiology and was assessed with two commonly used methodologies. There was a slower rate of rise and lower maximal amplitude of the melatonin rhythm in the AD group. DLMO varied by the method used to derive it. Using 3 pg/mL as threshold, no significant differences were found between the AD and HC groups. Using 2 standard deviations above the mean of the first three samples, the DLMO in AD occurred significantly later, 21:02?±?00:41?h, than in HC, 20:44?±?00:21?h (t?=??2.4, p?=?.02). Although melatonin in the AD group appears to have a slower rate of rise, using well-established criteria to assess the salivary DLMO did not reveal differences between AD and HC participants. Only when capturing melatonin when it is already rising was DLMO found to be significantly delayed by a mean 18?min in AD participants. Future circadian analyses on alcoholics should account for these methodological caveats. (Author correspondence: daconroy@med.umich.edu)     

Comparison of daily rhythms of oxygen metabolites and serum barrier to oxidation in domestic animals

The aim of this study was to compare the daily rhythm of reactive oxygen metabolites (hydroperoxides), and serum barrier to oxidation in five different mammal species. Five female Comisana sheep, five female Maltese goats, five female Bruna cows and five female Thoroughbred horses were housed separately in boxes equipped with an opening window and five female Blue Vienna rabbits were individually housed in metallic cages. All animals were kept under natural photoperiod and indoor environmental conditions. Blood samples were collected every 3 h over a 48 h period for the assessment of hydroperoxides, antioxidant barrier (Oxy-ads) and thiol-antioxidant barrier (SHp). A two-way for repeated measures ANOVA showed a significant effect of time of day and different species on all studied parameters. Daily rhythmicity of hydroperoxides, Oxy-ads and SHp were observed in all studied species. Nocturnal acrophases were observed in sheep, goats and rabbits, diurnal acrophases were observed in cattle and horse. We conclude that there is a synergism of the daily rhythms of hydroperoxides and anti-oxidant power in the different species studied, and that this synergism is typical of each species.     

Infrared methodologies for the assessment of skin temperature daily rhythm in two domestic mammalian species

To assess the accuracy of infrared methodologies for daily rhythm monitoring of skin temperature, five clinically healthy Italian Saddle gelding horses, and five not pregnant and not lactating Camosciata goats, were monitored every 4 h over a 48 h period. The horses were housed in individual boxes, while the goats in two indoor pens, under natural photoperiod and natural environmental temperature. In each animal, skin temperature was recorded with the use of a digital infrared camera and a non-contact infrared thermometer, in five regions: neck, shoulder, ribs, flank and croup. Recorded values were compared with the well-established daily rhythm of rectal temperature. Rectal temperature was recorded at the same time by means of a digital thermometer. In horses, a lower value of skin temperature was recorded using the infrared thermometer for the croup region compared to shoulder and flank; a lower value of skin temperature was recorded using thermography for the croup region compared to the shoulder. In goats, a lower value of skin temperature was recorded using the infrared thermometer for the croup region compared to the flank. In both species, higher values of rectal temperature were observed, compared to the temperature recorded at the skin regions using the other two methodologies. Cosinor rhythmometry showed a daily rhythm of rectal and skin temperature recorded using both methodologies in all the examined regions. General linear model (GLM) showed statistically significant effect of breed on all rhythmic parameters; of day of monitoring on amplitude; of site of recording (rectal vs skin regions) on mesor, amplitude and acrophase; and no effect of methodologies used. The results of this study show the differences in rhythmicity of various body regions temperature and their differences in comparison with daily rhythm rectal temperature. The use of infrared methodologies was inaccurate in assessing body core temperature, but its use could be considered for the evaluation of inflammation in the different body sites.     

The Circadian Rhythm of Body Temperature of the Horse

Rectal temperature of 10 female adult horses was recorded every 2 h for 10 consecutive days under a natural winter photoperiod (9 h of light and 15 h of darkness per day). A robust daily rhythm of body temperature was observed in all animals. The rhythm had a mean level of 38.3°C and a range of excursion of 1.0°C. Temperature started its daily ascent at dawn each day and reached a maximum 14 hours later. Body temperature of 5 of the horses was studied for 10 more days under constant illumination. The rhythm persisted under this condition, although with a slightly longer period of 24.2 h, which confirms the endogenous nature of the rhythm. Despite the fact that the body size of the horse is several orders of magnitude greater than that of rodents, the various parameters of the body temperature rhythm of the horse are similar to those of several species of rodents previously studied.     

Circadian rhythm gene expression and daily melatonin levels vary in athletes and sedentary males

The circadian rhythm is a 24-h cycle in which cells control metabolic and physiological processes throughout the day. In this study, we compared the expression patterns of major circadian rhythm-related genes: from blood of Bmal1, Ror-α, Cry1, Per2, Per1, and Nr1d1. In addition, changes in patterns of melatonin levels were observed in 16 subjects, eight males rugby players and eight males who did not exercise regularly. Blood was collected at 6:00, 10:00, 18:00, and 22:00. Bmal1, Ror-α, Cry1, Per2 (p < 0.001), Per1 (p < 0.01), and Nr1d1 (p < 0.05) genes related to circadian rhythm was higher in rugby players than in sedentary males. However, melatonin levels were higher in sedentary males than in rugby players (p < 0.05). These results indicate that long-term exercise in athletes can increase the expression of genes related to circadian rhythm and these may have an effect on daily melatonin levels as well.     

The daily rhythms of melatonin and free fatty acids in goats under varying photoperiods and constant darkness

The purpose of the study was to explore parallel and divergent features of the daily rhythms of melatonin and plasma free fatty acids (FFA) in goats exposed to different lighting conditions. From these features, we attempted to analyze whether the endogenous melatonin rhythm plays any role in the maintenance of the FFA rhythm. Seven Finnish landrace goats were kept under artificial lighting that simulated the annual changes of photoperiod at 60°N (longest photoperiod, 18 h; shortest, 6 h). The ambient temperature and feeding regimen were kept constant. Blood samples were collected 6 times a year at 2 h intervals for 2 d, first in the prevailing light-dark (LD) conditions and then after 3 d in constant darkness (DD). In LD conditions, the melatonin levels always increased immediately after lights-off and declined around lights-on, except in winter (18 h darkness), when the low daytime levels were restored clearly before lights-on. The FFA levels also displayed a consistent rhythmicity, with low levels at night and a transient peak around lights-on. In DD conditions, the melatonin profiles were very similar to those found in the habitual LD conditions, but the rhythm tended to advance. The FFA rhythm persisted also in DD, and the morning peak tended to advance. There was an overall parallelism between the two rhythms, with one significant exception. In winter in LD conditions, the morning rise in FFA levels coincided with lights-on and not with the declining phase of melatonin, whereas in DD conditions, the FFA peak advanced several hours and coincided with the declining phase of melatonin. From this finding and comparisons of the calculated rhythm characteristics, i.e., phase-shifts, phase differences, and correlations, we conclude that the daily rhythm of FFA levels is most probably generated by an endogenous oscillator, primarily adjusted by dawn, whereas the melatonin rhythm in this species is regulated by an oscillator primarily adjusted by dusk. The results did not exclude a modulatory effect of melatonin on the daily FFA profiles, but melatonin secretion, alone, does not explain the patterns sufficiently.     

Ring the Bell for Matins: Circadian Adaptation to Split Sleep by Cloistered Monks and Nuns

Cloistered monks and nuns adhere to a 10-century-old strict schedule with a common zeitgeber of a night split by a 2- to 3-h-long Office (Matins). The authors evaluated how the circadian core body temperature rhythm and sleep adapt in cloistered monks and nuns in two monasteries. Five monks and five nuns following the split-sleep night schedule for 5 to 46 yrs without interruption and 10 controls underwent interviews, sleep scales, and physical examination and produced a week-long sleep diary and actigraphy, plus 48-h recordings of core body temperature. The circadian rhythm of temperature was described by partial Fourier time-series analysis (with 12- and 24-h harmonics). The temperature peak and trough values and clock times did not differ between groups. However, the temperature rhythm was biphasic in monks and nuns, with an early decrease at 19:39?±?4:30?h (median?±?95% interval), plateau or rise of temperature at 22:35?±?00:23?h (while asleep) lasting 296?±?39?min, followed by a second decrease after the Matins Office, and a classical morning rise. Although they required alarm clocks to wake-up for Matins at midnight, the body temperature rise anticipated the nocturnal awakening by 85?±?15?min. Compared to the controls, the monks and nuns had an earlier sleep onset (20:05?±?00:59?h vs. 00:00?±?00:54?h, median?±?95% confidence interval, p?=?.0001) and offset (06:27?±?0:22?h, vs. 07:37?±?0:33?h, p?=?.0001), as well as a shorter sleep time (6.5?±?0.6 vs. 7.6?±?0.7?h, p?=?.05). They reported difficulties with sleep latency, sleep duration, and daytime function, and more frequent hypnagogic hallucinations. In contrast to their daytime silence, they experienced conversations (and occasionally prayers) in dreams. The biphasic temperature profile in monks and nuns suggests the human clock adapts to and even anticipates nocturnal awakenings. It resembles the biphasic sleep and rhythm of healthy volunteers transferred to a short (10-h) photoperiod and provides a living glance into the sleep pattern of medieval time. (Author correspondence: isabelle.arnulf@psl.aphp.fr)     

The daily rhythms of melatonin and free fatty acids in goats under varying photoperiods and constant darkness

The purpose of the study was to explore parallel and divergent features of the daily rhythms of melatonin and plasma free fatty acids (FFA) in goats exposed to different lighting conditions. From these features, we attempted to analyze whether the endogenous melatonin rhythm plays any role in the maintenance of the FFA rhythm. Seven Finnish landrace goats were kept under artificial lighting that simulated the annual changes of photoperiod at 60°N (longest photoperiod, 18 h; shortest, 6 h). The ambient temperature and feeding regimen were kept constant. Blood samples were collected 6 times a year at 2 h intervals for 2 d, first in the prevailing light‐dark (LD) conditions and then after 3 d in constant darkness (DD). In LD conditions, the melatonin levels always increased immediately after lights‐off and declined around lights‐on, except in winter (18 h darkness), when the low daytime levels were restored clearly before lights‐on. The FFA levels also displayed a consistent rhythmicity, with low levels at night and a transient peak around lights‐on. In DD conditions, the melatonin profiles were very similar to those found in the habitual LD conditions, but the rhythm tended to advance. The FFA rhythm persisted also in DD, and the morning peak tended to advance. There was an overall parallelism between the two rhythms, with one significant exception. In winter in LD conditions, the morning rise in FFA levels coincided with lights‐on and not with the declining phase of melatonin, whereas in DD conditions, the FFA peak advanced several hours and coincided with the declining phase of melatonin. From this finding and comparisons of the calculated rhythm characteristics, i.e., phase‐shifts, phase differences, and correlations, we conclude that the daily rhythm of FFA levels is most probably generated by an endogenous oscillator, primarily adjusted by dawn, whereas the melatonin rhythm in this species is regulated by an oscillator primarily adjusted by dusk. The results did not exclude a modulatory effect of melatonin on the daily FFA profiles, but melatonin secretion, alone, does not explain the patterns sufficiently.     

Asymbiotic seed germination and in vitro seedling development of <Emphasis Type="Italic">Cyrtopodium punctatum</Emphasis>: a propagation protocol for an endangered Florida native orchid

Cyrtopodium punctatum Lindley is an endangered epiphytic orchid restricted in the United States to southern Florida. Due to its ornamental value, the species was extensively collected from the wild during the past 100 years. Today, only a few plants remain in protected areas. As part of a conservation plan for the species, procedures for asymbiotic seed germination were developed. Five asymbiotic orchid seed germination media (PhytoTechnology Orchid Seed Sowing Medium, Knudson C, Malmgren Modified Terrestrial Orchid Medium, Vacin &; Went Modified Orchid Medium, and ½-strengh Murashige &; Skoog) were examined for their effectiveness in promoting seed germination and protocorm development under a 16/8 h L/D photoperiod and dark (0/24 h L/D). The influence of photoperiod on growth and development was also examined. Seeds were germinated under a 16/8 h, 12/12 h, 8/16 h L/D photoperiod, at 25 ± 3°C and allowed to develop in vitro for 10 weeks. After 10 weeks, developing seedlings were transferred to Sigma Phytatrays and returned to their assigned photoperiod treatments for continued seedling development for an additional 15 weeks. Highest germination occurred in 0/24 h L/D on PhytoTechnology Orchid Seed Sowing Medium and seedlings displayed more advanced development when cultured under 16/8 h L/D photoperiod after 15 weeks in Phytatrays. Thirty-five week old seedlings potted in coconut husk growing medium exhibited 90% survival following 5 weeks acclimatization to greenhouse conditions. This asymbiotic seed germination protocol for C. punctatum will facilitate future reintroduction projects involving this endangered species.     

Endogenous activity rhythm in Talitrus saltator,Britorchestia brito (Crustacea,Amphipoda) and Tylos europaeus (Crustacea,Isopoda) from Barkoukech Beach (Tabarka,Tunisia)

The locomotor activity rhythm of three supralittoral crustacean species, Tylos europaeus (terrestrial isopod) and Britorchestia brito and Talitrus saltator (amphipod, Talitridea), was investigated in Barkoukech Beach. The rhythm was monitored in individual animals in spring under continuous darkness and constant temperature. Results revealed that whatever the species, actograms and mean activity curves showed that specimens of T. europaeus and T. saltator concentrated their activity during the subjective night; whereas, B. brito concentrated its activity during the subjective day. The three species exhibited a circadian locomotor rhythm with mean circadian period, respectively, equal to 24h41′ ± 0h44′, 24h46′ ± 0h55′ and 25h21′ ± 1h16′. The most stable locomotor rhythm was found in T. saltator (0.444 ± 0.246). Furthermore, individuals of T. europaeus were significantly more active (10h47′ ± 2h52′) than the two amphipods. Differences in the activity patterns of the three species will be discussed as an adaptative strategy to respond to interspecific competitive interactions.