Maintenance of permanent sexual activity throughout the year in seasonal bucks using short photoperiodic cycles in open barns.

Seasonality of reproductive activity in rams and bucks is the major constraint in temperate and subtropical zones. Rapid alternation between 1 month of short days and 1 month of long days (LD) over three years in lightproof buildings eliminates this seasonality. We examined if this would also work in open barns, using only supplementary light. Over two years, one group of bucks (n = 7) was subjected to alternate 1 month of LD and 1 month of permanent light (LD-LL) and another group (n = 7) to alternate 1 month of LD and 1 month of natural light (LD-NL). A simultaneous control group, used for both experiments (CG1, n = 6; CG2, n = 6), remained under natural photoperiod. BW, testis weight (TW), plasma testosterone (T) and cortisol (C) were evaluated in all bucks. CG1 and CG2 bucks showed identical dramatic seasonal variations in BW (stable or decreasing in summer), TW (from 85 ± 12 g in February to 127 ± 7 g in July) and T (from 2.7 ± 1.2 ng/mL in January-April to 24.3 ± 3.2 ng/mL in June-October). By contrast, BW of LD-LL and LD-NL bucks increased regularly during the experiment. From 5 and 9 months after the experiment onset, LD-LL and LD-NL bucks, respectively, maintained constant TW of 115 ± 5 g until the experiment end. After the first 3 months <5 ng/mL, T of LD-LL bucks remained constant (5-10 ng/mL) until the experiment end. By contrast, T of LD-NL bucks showed four periods of low (<5 ng/mL) and two periods of high concentrations (18.1 ± 2.6 and 11.9 ± 3.4 ng/mL). Plasma C remained low (5-8 ng/mL) and did not change with group or light treatment. These results show for the first time in any seasonal photoperiodic species that it is possible to maintain the sexual activity of males all year round in open buildings using alternating periods of LD and LL. By contrast, return to NL instead of LL every other month does not prevent seasonality in T concentration. These results raise interesting questions about the photoperiodic control of neuroendocrine regulation of seasonal sexual activity and suggest that these treatments can be used to manage males in open barns in farms and in artificial insemination centres. (Spanish and French versions of the full text are available as Supplementary Materials S1 and S2).

Continuous presence of females in estrus does not prevent seasonal inhibition of LH and androgen concentrations in bucks.

In male goats, being in permanent visual contact with females in estrus does not prevent seasonal variation in certain endocrine hormone levels and sexual activities. In this study, we tested whether continuous and full contact with females in estrus prevented seasonal endocrinological variation in bucks. In 1 experiment (Exp. 1), we verified that the sudden introduction of goats in estrus increased the plasma concentrations of androgen in bucks during the nonbreeding season under our experimental conditions. In another experiment (Exp. 2), we tested the ability of estrous goats to prevent seasonal inhibition of LH and androgen secretions in bucks kept in permanent and full contact with them. In Exp. 1, 3 groups of bucks (n = 5 in each group) were isolated from females from the months of July to January. On January 27, one group continued being isolated from females; a second group was exposed to ovariectomized, untreated goats; and a third group was exposed to ovariectomized goats with induced estrus. Plasma androgen concentrations were determined every 2 h from 8 h before to 8 h after the introduction of females. The introduction of estrus-induced goats significantly increased androgen concentrations, which were higher than in the isolated bucks, as well as in those exposed to untreated goats (P < 0.05). In Exp. 2 (n = 5 per group), one group of bucks was isolated from females from October to July, whereas two other groups remained in contact with ovariectomized goats, either untreated or regularly induced to estrus. In the three groups of bucks, plasma concentrations of LH were determined once during the months of October, February, March, and June, whereas androgen concentrations were determined weekly from October to July. The mean plasma LH and androgen concentrations were low and did not differ among the groups of bucks during the normal seasonal period of sexual inactivity (P > 0.05). We conclude that full contact and sexual interactions with estrus-induced goats failed to stop the seasonality of LH and androgen plasma concentrations of bucks, although bucks could respond to the introduction of females by acute increases in plasma LH and androgen.

Highly precocious activation of reproductive function in autumn-born goats (Capra hircus) by exposure to sexually active bucks.

Goats are seasonal breeders with the main cue controlling the timing of breeding season being photoperiod. Hence, the season of birth impacts puberty onset: spring-born goats reach puberty in autumn, at 7 mo of age, whereas autumn-born goats reach puberty at 1 yr during the next reproductive season. The aim of this study was to determine whether exposure of autumn-born young females to sexually active males could counteract the delay in puberty onset observed in autumn-born goats. Females exposed to sexually active males (n = 8) reached puberty earlier than isolated females (n = 8), with exposed females ovulating at a mean age of 3.5 mo. To our knowledge, such precocious puberty onset obtained through social stimulation has never been described in the literature. Moreover, those exposed females exhibited estrus behavior for most ovulations. Our results indicate that in goats born out of season, exposure to sexually active bucks is a really efficient approach to induce early puberty, suggesting that social interactions could have a crucial impact on the regulation of pubertal transition.

Sexually active bucks counterbalance the seasonal negative feedback of estradiol on LH in ovariectomized goats.

We showed previously that the permanent presence of bucks rendered sexually active by photoperiodic treatments, thereafter called photostimulated bucks, prevents the occurrence of seasonal anovulation; also, the introduction of these sexually active bucks induces ovulations during seasonal anestrus. Here, we studied the response of ovariectomized goats bearing 12-mm subcutaneous implants filled or not with estradiol to sexually active males to determine (1) whether the permanent presence of such bucks prevents the decrease of LH despite the presence of a negative feedback by estradiol mimicking that of seasonal anestrus (experiment 1) and (2) whether the introduction of photostimulated bucks increases the plasma LH concentrations in spite of this negative feedback (experiment 2). In experiment 1, one group of goats remained in contact with sexually active bucks, whereas the other group remained in contact with control bucks. Plasma LH concentrations were high and did not differ with time or between groups of females from November to February (P > 0.05), when both types of bucks were sexually active. Afterward, in goats in contact with control and sexually inactive bucks, LH concentrations decreased from March (P ≤ 0.01) and remained low until May, whereas LH levels remained high from March to May in goats in contact with the photostimulated bucks (P > 0.05). In experiment 2, 2 groups of females bearing empty subcutaneous implants, and 2 groups of goats bearing subcutaneous implants filled with estradiol, were exposed to control or photostimulated bucks. Plasma LH concentrations did not increase in goats bearing empty implants, when exposed to control or photostimulated bucks (from 2.01 ± 0.26 to 1.98 ± 0.31 ng/mL, and from 2.45 ± 0.29 to 2.42 ± 0.21 ng/mL respectively; P > 0.05). In contrast, plasma LH concentrations increased from 0.97 ± 0.41 to 2.80 ± 0.62 ng/mL in goats exposed to the photostimulated bucks and bearing estradiol implants (P < 0.05). Thus, the permanent presence of sexually active bucks prevented the decrease of plasma LH concentration in OVX + E2 goats during the seasonal anestrus, and the introduction of the photostimulated bucks increased the plasma LH concentrations in OVX + E2 goats during the seasonal anestrus. Therefore, we conclude that in both cases, the photostimulated bucks are able to reduce or counterbalance the seasonal negative feedback of estradiol on LH secretion.

Continuous light after 2 months of long days stimulates ram testis volume and increases fertility in spring.

Seasonal reproduction is one of the major biotechnical and economic constraints of sheep production in temperate latitudes. Treatments using extra light followed by melatonin implants have been used satisfactorily in open barns, farms and artificial insemination centres to produce out-of-season sexual activity in rams. The aim of the present study is to explore the possibility of replacing melatonin implants with continuous light (LL), which was recently shown to increase LH secretion similar to melatonin and/or pinealectomy. Four experiments during 4 consecutive years were conducted in 'Ile-de-France' rams. In each study, one group was systematically exposed to permanent light after a first photoperiodic treatment of 60 long days (LD-LL) during the winter and compared with various other control groups subjected either to a natural photoperiod or the classical LD-melatonin treatment. As expected, blood nocturnal melatonin secretion was suppressed by LL. In all four experiments, LL treatment produced a highly significant and robust increase in ram testicular volume in the spring compared with the testicular volume of control rams or of that of treated rams at the end of the LD. For the two experiments in which fertility was tested, fertility after hand-mating was significantly higher in LD-LL rams than in control rams (76% v. 64%). Therefore, permanent light after an LD treatment may be an interesting alternative to LD-melatonin treatment to induce out-of-season sexual activity in rams.

Melatonin from cerebrospinal fluid but not from blood reaches sheep cerebral tissues under physiological conditions.

The pineal gland secretes melatonin (MLT) that circulates in the blood and cerebrospinal fluid (CSF). We provide data to support the hypothesis that, in sheep and possibly in humans, only the CSF MLT, and not the blood MLT, can provide most of MLT to the cerebral tissue in high concentrations, particularly in the periventricular area. The MLT content of sheep brain, our chosen animal model, was found in significant concentration gradients oriented from the ventricle (close to the CSF) to the cerebral tissue, with concentrations varying by a factor of 1-125. The highest concentrations were observed close to the ventricle wall, whereas the lowest concentrations were furthest from the ventricles (407.0 ± 71.5 pg/ml compared to 84.7 ± 5.2 pg/ml around the third ventricle). This concentration gradient was measured in brain tissue collected at mid-day and at the end of the night. Nocturnal concentrations were higher than daytime concentrations, reflecting the diurnal variation in the pineal gland. The concentration gradient was not detected when MLT was delivered to the brain via the bloodstream. The diffusion of MLT to cerebral tissues via CSF was supported by in vivo scintigraphy and autoradiography. 2-[(123)I]-MLT infused into the CSF quickly and efficiently diffused into the brain tissues, whereas [(123)I]-iodine (control) was mostly washed away by the CSF flow and [(123)I]-bovine serum albumin remained mostly in the CSF. Taken together, these data support a critical role of CSF in providing the brain with MLT.

Functional characterization of polymorphic variants for ovine MT1 melatonin receptors: possible implication for seasonal reproduction in sheep.

In seasonal breeding species, the gene encoding for the melatonin MT(1) receptor (oMT(1)) is highly polymorphic and numerous data have reported the existence of an association between an allele of the receptor and a marked expression of the seasonality of reproduction in ewes. This allele called "m" (previously named "-" allele) carries a mutation leading to the absence of a MnlI restriction site as opposed to the "M" allele (previously named "+" allele) carrying the MnlI restriction site (previously "+" allele). This allows the determination of the three genotypes "M/M" (+/+), "M/m" (+/-) and "m/m" (-/-). This mutation is conservative and could therefore not be causal. However, it is associated with another mutation introducing the change of a valine to an isoleucine in the fifth transmembrane domain of the receptor. Homozygous "M/M" and "m/m" animals consequently express structurally different receptors respectively named oMT(1) Val(220) and oMT(1) Ile(220). The objective of this study was to test whether these polymorphic variants are functionally different. To achieve this goal, we characterized the binding properties and the transduction pathways associated with both variants of the receptors. Using a pharmacological approach, no variation in binding parameters between the two receptors when transiently expressed in COS-7. In stably transfected HEK293 cells, significant differences were detected in the inhibition of cAMP production whereas receptors internalization processes were not different. In conclusion, the possibility that subtle alterations induced by the non conservative mutation in "m/m" animals might modify the perception of the melatoninergic signal is discussed in the context of melatonin action.

Differential estradiol requirement for the induction of estrus behavior and the luteinizing hormone surge in two breeds of sheep.

For a better understanding of the mechanisms that lead to the preovulatory GnRH/LH surge and estrus behavior, the minimum estradiol (E) requirements (dose and duration) to induce each of these events were determined and compared between two breeds of ewes having either single (Ile de France) or multiple (Romanov) ovulations. The ewes were initially studied during a natural estrus cycle, and were then ovariectomized and run through successive artificial estrus cycles. For these artificial cycles the duration and amplitude of the follucular phase E increase were manipulated by E implants. Under all conditions, the onset of estrus behavior was similar in the two breeds, although its duration was longer in Romanov ewes. While a moderate E signal (6 cm for 12 h) induced an LH surge in 10/10 Ile de France ewes, a larger E signal (12 cm for 12 h) was minimally effective in Romanov ewes (4/10). Additional studies revealed that a small E signal (3 cm for 6 h) induced full estrus behavior in all Romanov ewes but was completely ineffective in Ile de France animals (0/10). Higher doses and mostly longer durations of the E signal (12 cm for 24 h) were required to induce a surge in all the Romanov ewes. These results demonstrate a clear difference in the E requirement for the induction of estrus behavior and the LH surge between breeds of ewes that have different ovulation rates. These data provide compelling evidence that, in one breed, the neuronal systems that regulate both events require different estrogen signals.

Importance of learning in the response of ewes to male odor.

Exposure of anestrous ewes to a ram or its odor results in the activation of the luteinizing hormone (LH) secretion leading to reinstatement of cyclicity in most females. Sexual experience and learning have been suggested as important factors to explain the variability of the female responses. In experiment 1, we compared the behavioral and endocrine responses of four groups of anestrous females that differed in age (young or adult) and previous exposure to males [naive (no exposure) or experienced (courtship behavior for young and numerous mating for adults)]. Age did not seem to affect the LH response to males or their odor. In contrast, sexual experience was a critical factor: the proportion of females exhibiting an LH response to male odor was significantly higher in experienced than in naive ewes. Sexual experience affected the response to male odor, but did not have an effect on responses to the male himself. A second experiment investigated whether the LH response to male odor could result from an associative learning process. Accordingly, we tested the effectiveness of a conditioned stimulus (lavender odor) previously associated with the male, in inducing the endocrine response. The results indicate that the odor of lavender activated LH secretion only in ewes that have been previously exposed to scented males. This demonstrates that ewes are able to learn the association between a neutral odor and their sexual partner.

Inactivation of the olfactory amygdala prevents the endocrine response to male odour in anoestrus ewes.

Our aim was to study the role of the olfactory amygdala (medial and cortical nuclei) and the ventromedial nucleus of the hypothalamus (VMN) in the ability of the male odour or live males to induce a release of luteinizing hormone in anoestrus ewes. To achieve this, we temporarily blocked the activity of these structures by localized retrodialysis administration of the anaesthetic lidocaine. The effect of ram odour on the secretion of luteinizing hormone was completely blocked by inactivation of the cortical nucleus of the amygdala. In contrast, inactivation of part of the accessory olfactory system (the medial nucleus of the amygdala or the VMN) had no effect. In the presence of the male, lidocaine never impaired the endocrine response of the ewes. These results show that modulation of reproduction by the sexual partner even through pheromonal cues does not occur via the direct circuit of the accessory system. On the contrary, the cortical nucleus of the amygdala is absolutely necessary for the treatment of and/or the response to the male olfactory signal but this structure can be bypassed when other sensory cues are available.

Role of experience in the neuroendocrine control of ewes' sexual behavior.

We assessed the role of learning in the expression of female sexual behavior and evaluated the relative importance of age versus experience. Two studies were conducted with ovariectomized ewes submitted to steroid treatment that mimicked an estrus cycle. We compared behavioral (experiments 1 and 2), neurochemical (experiment 1), and endocrine (experiment 2) responses of sexually naive young and adult ewes versus sexually experienced adults when exposed to males. In a third study, we compared their performance in an instrumental learning test and the extent to which it was affected by stress. These experiments showed that proceptivity is affected both by age and sexual experience. In experiment 1 only experienced adults were proceptive and displayed an increase in hypothalamic norepinephrine. By the second estrus cycle (experiment 2) naive adults performed similarly to experienced adults but proceptive behavior was still inferior in young ewes. Receptivity was also different between groups but affected more by age than by sexual experience. All ewes mated during the first interaction with a male, although males' latency to ejaculation was shorter with experienced females than naive adults or naive young. Young ewes found food as readily as adults in experiment 3 but were more affected by stress. Together, these experiments show that both experience and age influence sexual activity and that sensitivity to stress may also be involved. This may contribute to the deficient reproductive performance that is often observed in young female mammals.

Sequential role of e2 and GnRH for the expression of estrous behavior in ewes.

Preovulatory GnRH secretion in ewes, measured in portal blood and cerebrospinal fluid, starts at the time of the LH surge, approximately 4 h after the onset of estrous behavior, and lasts as long as receptivity (36-48 h), which is much longer than the LH surge. This study tested the hypothesis that the extended GnRH secretion is involved in the maintenance of receptive behavior, prolonging the initial triggering effect of E2. Ovariectomized ewes were subjected to artificial estrous cycles and infused intracerebroventricularly either with a water soluble GnRH antagonist (Teverelix, Exp 1 and 2) or GnRH (Exp 3 and 4) after preovulatory E2 challenges of various intensity. The GnRH antagonist infused for 20 h (0.5 mg/ml, flow rate 3 microl/min) following a treatment with 2 x 30-mm E2 implants for 24 h (Exp 1) significantly reduced receptivity 36-48 h post E2 compared with vehicle infusion. By contrast, when the GnRH antagonist was infused with E2 implants still present (Exp 2: E2 for 48 h, GnRH antagonist given 24-44 h after E2 insertion, n = 14) receptivity was not affected. Administration of GnRH (0.5 mg/ml, flow rate 3 microl/min) when receptivity began to decline (Exp 3: 30-48 h after a 6-h 2 x 30-mm E2 implants n = 12) resulted in significantly higher receptivity scores at 48 and 52 h post E2 in GnRH treated animals compared with vehicle treated. GnRH infusion of ewes under subthreshold E2 treatment (Exp 4: GnRH 6-24 h after implantation of 1 x 30-mm E2 for 3 h, n = 12 in a cross-over design) significantly increased their receptivity compared with vehicle administration at 18 and 24 h post E2 insertion, but receptivity remained lower than when induced by high doses of E2. Our results demonstrate for the first time that GnRH is involved in the control of receptivity in a ruminant species and suggest that in the cycling ewe the sustained preovulatory GnRH secretion plays a physiological role in extending the duration of estrous behavior. They also indicate that it is possible to dissociate a direct effect of E2 on estrous behavior from its effect via stimulation of GnRH secretion.

The onset of increased melatonin secretion after the onset of darkness in sheep depends on the photoperiod.

In sheep, melatonin secretion occurs rapidly after the onset of darkness, but the interval fluctuates according to different authors. The aim of this study was to determine this interval in sheep subjected to a long or a short photoperiod. Blood samples were taken at the right jugular vein every 100 s for 1 hr after the onset of darkness. The experiment was repeated on three consecutive days either in long (LD 16:8) or in short photoperiod (LD 8:16) on the same animals. Melatonin secretion was found to begin within 11 min under long photoperiod and 20 min under short period. It can be concluded that the onset of melatonin secretion depends on the duration of the photoperiod.

Day/night variation of tryptophan hydroxylase and serotonin N-acetyltransferase mRNA levels in the ovine pineal gland and retina.

In mammals, the photoperiodic information, received by the retina, is transmitted to the pineal gland. In both organs, melatonin is produced and functions as a neurohormone giving temporal information to the organism. A four-step enzymatic pathway, involving in particular the tryptophan hydroxylase (TPOH), the rate-limiting enzyme in serotonin synthesis, and the serotonin N-acetyltransferase (NAT) that converts serotonin to N-acetylserotonin, allows the synthesis of melatonin. Many studies on melatonin synthesis modulation have focused on the enzyme NAT, but the regulation of TPOH is less well understood. We report here a quantitative study, using a reverse transcription polymerase chain reaction (RT-PCR) analysis, of the nycthemeral expression of TPOH and NAT mRNAs in the ovine retina and pineal gland. In both organs, we show a nocturnal increase in mRNA levels of the two enzymes. suggesting a role of transcriptional mechanisms in the regulation of melatonin synthesis. However, the amplitude of the observed increase in TPOH and NAT mRNAs expression can not entirely explain the 7-fold nocturnal increase in the plasma melatonin level. Our results suggest that, in the sheep, post-transcriptional mechanisms might also be involved in the day/night modulation of melatonin production.

Melatonin secretion in rams maintained in constant darkness depends on the timing of a single 1-hour light pulse given the previous night.

Previous data have demonstrated that a single 1-hr light pulse at night affects the secretion of melatonin in the ram if it was given at the appropriate time. This paper reports the effect on melatonin secretion of a 1-hr light pulse given at two different times at night to two groups of rams kept in constant darkness the day following light application. It appears that the animals were able to remember the light pulse if it was given 12 hr but not 9 hr after the lights were turned off. This memory could possibly be stored in the suprachiasmatic nucleus as reported recently in the rat.

Pineal microdialysis of the melatonin in conscious sheep: methodology, application to a diurnal rhythm and effect of isoproterenol.

This paper describes the development of a new technique to measure melatonin contents in the pineal gland of moving sheep: the microdialysis. A dialysis probe was used to collect extracellular fluid in the sheep pineal gland, but also to inject directly into it different drugs such as isoproterenol at a very low concentration. The probe was implanted the day before the beginning of the experiment in order to obtain low levels of melatonin. This technique makes it possible to measure melatonin in the dialysate and plasma of rams submitted to 8L:16D. No melatonin either in the dialysate or in the plasma was found during the light phase. Shortly after lighting off, the melatonin concentration increased in the dialysate and plasma and remained stable during the dark phase. Melatonin concentrations began to decrease before lighting on and no detectable levels were found during the following light phase. The secretion of melatonin is, at least, under adrenergic regulation. Local infusion of isoproterenol (90 microliters at 10(-6) M), an agonist of beta adrenergic receptor, through the probe, increased melatonin levels during 2 h, even when infusions were repeated 3 times. This demonstrates the presence of beta adrenergic receptors. The technique presented in this paper could be of considerable interest for studying sheep pineal gland and its main secretion, melatonin, for example during diurnal rhythms or for studying its regulation.

Rhythmic secretion of melatonin by the superfused pike pineal organ: thermo- and photoperiod interaction.

In the pineal organ of the pike (Esox lucius, teleost), the rhythmic production of melatonin by the photoreceptor cells is governed by a population of endogenous circadian oscillators, synchronized and entrained by the light/dark (L/D) cycle. Production of melatonin is inhibited by light and highly increased in the dark. In the present study, isolated and superfused pike pineals were exposed to a 24-hour temperature cycle of high (10 degrees C) or low (4 degrees C) amplitude, either under L/D, or under constant darkness. Under L/D, photoperiod is the dominant entraining stimulus for the melatonin secretion rhythm. It was high during the dark phase and low during the light phase, either under cold light/warm dark or under warm light/cold dark conditions. Under a warm light/cold dark cycle, the amplitude of the melatonin rhythm was reduced. In pineals cultured at 10 degrees C, a 20 degrees C temperature pulse potentiated or reduced the night-induced rise in melatonin production when applied, during the dark or during the preceding light phase, respectively. Under constant darkness, high- or low-amplitude temperature cycles could synchronize the rhythmic production of melatonin, which peaked with the high temperature. However, the shape of the oscillation could appear modified under warm subjective-L/cold subjective-D, depending on the experimental conditions. Finally, the rhythmic release of melatonin synchronized by a temperature cycle under constant darkness was no longer observed after removal of the external zeitgeber. The present study provides direct evidence that the pineal acts as a photothermotransducer.

Circadian and seasonal rhythms of melatonin production in mules (Equus asinus x Equus caballus).

The present study describes the patterns of melatonin production in the mule (Equus asinus x Equus caballus). Blood was sampled hourly for 24 h from eight mule mares in spring and fall. The data obtained show the presence of a circadian rhythm of production of melatonin, with highest values during the dark phase both in spring and fall. In fall the nightly rise of melatonin production begins earlier in the dark phase and reaches higher quantitative levels than in spring. The morning decline of melatonin production is similar in the two seasons. Maximal levels of nightly melatonin production in the mule are about 10 x higher than those described in the horse. The results reported here indicate the persistence of brain structures able to receive and transduce environmental signals in the mule, a genetically sterile mammalian hybrid.

Entrainment of melatonin rhythms in rams by symmetrical light-dark cycles of different period length.

The objective of this study was to investigate the entrainment of melatonin rhythms in rams using symmetrical light-dark cycles of different period length. Five groups of six Ile de France rams were kept in 12L:12D for 7 weeks and then (i) 12L:12D, (ii) 11L:11D, (iii) 10L:10D, (iv) 13L:13D and (v) 14L:14D for a further 3 weeks. Environmental factors other than the light dark cycle were not controlled. The onset and offset of the plasma melatonin rhythm in DD after 3 weeks of the respective light treatments was assessed for 48 hr, immediately after transferring to DD. The duration of secretion in DD was positively related to the length of the previous dark phase. The phase of the melatonin rhythm with respect to the anticipated dark phase suggested entrainment with no change in phase-relationship to the zeitgeber by 12L:12D and 13L:13D. Entrainment with a phase-delay or a phase-advance was apparent after 11L:11D and 14L:14D, but the individual rhythms were not all synchronized with respect to each other after 10L:10D. Activity recordings for 2-3-week periods during 12L:12D, 10L:10D and 14L:14D all showed a major 24-hr component at all times, with activity during the light phase in 12L:12D. It appears that melatonin may be readily desynchronized from overt activity-rest cycles in sheep. The upper and lower entrainment limits are probably greater than 28 hr and close to 20 hr cycles, respectively.