Synthesis and anthelmintic activity of 3'-benzoylurea derivatives of 6-phenyl-2,3,5,6-tetrahydroimidazo[2,1-b]thiazole.

Reaction of 3-amino derivatives of the nematocides tetramisole and levamisole with variously substituted benzoylisocyanates gave a series of benzoylureas I which were tested for activity against helminths and ectoparasites. Compounds bearing 2,6-difluoro and 4-trifluoromethyl substituents had potent nematocidal activity in both mice and sheep. No antiectoparasitic activity was observed.

Synthesis and biological activity of a series of diaryl-substituted alpha-cyano-beta-hydroxypropenamides, a new class of anthelmintic agents.

A series of alpha-cyano-beta-hydroxypropenamides was prepared and tested for anthelmintic activity. alpha-Cyano-beta-hydroxy-N-[4- (trifluoromethyl)phenyl]-3-[4-(trifluoromethyl)phenyl]propenamide (1) showed good activity against the nematode Nematospirodes dubius in a mixed parasite infection in mice; several of the analogues were also effective against the cestode Hymenolepis nana. In sheep trials, 1 caused 100% reduction of the hematophagous nematode Haemonchus contortus after a single dose of 20 mg/kg but did not show satisfactory control of Trichostrongylus colubriformis or Ostertagia circumcincta. Against the liver fluke Fasciola hepatica, 1 suppressed egg production but only temporarily, suggesting that the adult flukes were not eliminated. Mechanism of action studies on 1 using Ascaris mitochondria showed it to be an uncoupler of oxidative phosphorylation.

Changes in plasma concentrations of LH, FSH and prolactin in ewes receiving melatonin and short-photoperiod treatments to induce early onset of breeding activity.

Breeding activity was similarly advanced in ewes given continuous (s.c. implant) or timed (oral dose at 15.30 h) melatonin treatments or subjected to a short (8 h light: 16 h darkness) artificial photoperiod. Treatments commenced in mid-June and were terminated in mid-November. Weekly and serial blood samples were collected before and after treatments commenced, to ascertain the effects on plasma prolactin, LH and FSH concentrations. In addition, serial blood samples were collected for 24 h plasma prolactin and melatonin estimations before and after cessation of the treatments. Plasma prolactin levels were significantly reduced immediately following the start of the melatonin (implant and oral) and short-photoperiod treatments but 'rebounded' to levels greater than control values. The normal seasonal (spring) rise in plasma prolactin was noted in the following year. Before the onset of breeding activity, mean plasma LH and FSH concentrations and LH pulse frequency did not change following any of the treatments. The 24-h plasma melatonin profile accurately reflected the various applied treatments but had re-entrained to the prevailing (natural) photoperiod 1 week after termination of the treatments. There were no significant group differences in 24-h plasma prolactin levels 1 week before or 1 and 11 weeks after the treatments had ceased. Such treatments, although successfully advancing the onset of breeding activity and modifying the seasonal plasma prolactin rhythm, were not manifested through any apparent change in peripheral LH or FSH.

Effects of various melatonin treatments on plasma prolactin concentrations in the ewe.

Ewes were treated with s.c. implants of melatonin in mid-April, mid-May and mid-June. From mid-June, other animals were given oral doses of melatonin daily at 16.30 h and another group was maintained under a short (8 h light:16 h darkness) artificial photoperiod (lights out 16.30 h). Serial blood samples were taken from all animals in June and July. Plasma prolactin concentrations were significantly reduced in ewes treated in May and June (implant, oral and photoperiod treatments) but not in those treated in April. After treatment in June, prolactin levels were significantly suppressed after 7 days of oral and implant melatonin therapy, and after 28 days of a short artificial photoperiod. Melatonin treatment appeared more efficient than an artificial photoperiod in reducing plasma prolactin concentrations.

Induction of early seasonal sensitivity to melatonin in Suffolk-Cross ewes.

Anoestrous Suffolk-Cross ewes can be induced into early seasonal ovarian activity by administration of melatonin at the appropriate time of day or by melatonin implants. This treatment is successful if commenced in June, but not earlier in April or May and suggests that a critical period of long days may be necessary before artificial short-day melatonin profiles act as winter time-cues. We have investigated whether the lack of sensitivity to melatonin in April could be overcome in ewes in which breeding activity had been artificially moved forward the previous season. The results indicate that this was indeed the case and that the breeding season in untreated ewes which also previously experienced an early induced breeding season reverted to the normal timing for the Suffolk-Cross breed.

Use of an intraruminal soluble glass bolus containing melatonin for early lamb production.

The use of melatonin to advance the onset of seasonal oestrus is potentially useful for early lamb production. A number of delivery systems for melatonin have been developed and this study evaluates an intraruminal soluble glass bolus under field conditions. Anoestrous, non-lactating mule and Dorset cross mule ewes were treated in late June with two types of bolus of different solubilities (melatonin 1, 'slow release' and melatonin 2, 'extra slow release'). Rams were introduced in mid-August for a target lambing pattern in mid-January. The percentage pregnancy/lambing rates were 80/78, 97/92 and 100/100 in the control, melatonin 1 and melatonin 2 groups, respectively. The number of lambs born per ewe put to the ram was significantly increased by treatment with melatonin 2 (P less than 0.05) from 1.17 to 1.64 in the mule ewes and 1.64 to 1.72 in the Dorset cross mule ewes. The melatonin containing soluble glass bolus is a novel and convenient method of reducing the number of barren ewes in an early lambing flock.

Anthelmintic resistance in Australian sheep nematode populations.

The resistance status of gastro-intestinal nematodes to anthelmintics was evaluated on 881 sheep farms throughout Australia during 1991-92. Resistance was shown to be widespread. Overall, 85% of farms had sheep infected with nematodes resistant to benzimidazole, 65% to levamisole and 34% to combination (benzimidazole+levamisole) products. Resistance to ivermectin was not detected. On only 9% of farms did all anthelmintic groups reduce egg counts by greater than or equal to 95%. The culture of faeces from untreated sheep showed Telodorsagia circumcincta, Trichostrongylus spp, Chabertia ovina and Haemonchus contortus to be the principal species. The nature and prevalence of resistance was not significantly correlated with stocking rate. However, resistance to combination products was almost twice as prevalent on farms in areas with an average annual rainfall of greater than 500 mm.

Response of pre-pubertally castrated rams and ewes to artificial photoperiod--changes in plasma LH and prolactin.

Castrate rams and ovariectomized ewes were maintained in the presence of entire rams and ewes and subjected to successive periods of alternating 6 h light:18 h darkness ('short' days) and 18 h light:6 h darkness ('long' days) preceded by a period of 12 h light:12 h darkness ('constant' light days). Plasma concentrations of LH and prolactin were measured in the castrate animals in order to determine how LH and prolactin secretion responded to the artificial light regime and corresponding periods of elevated or depressed testicular and ovarian activity in the entire rams and ewes. There was no variation in mean plasma LH concentrations or LH pulse frequency with either the changes in photoperiod or the phases of gonadal activity in the entire animals. However, there was a highly significant (P less than 0.001) relationship between prolactin secretion and the artificial photoperiod in both castrate groups with high and low levels coinciding with long and short days respectively. In addition, there was a marginally significant (P less than 0.1) relationship between prolactin secretion in the castrate ram and the stage of testicular activity in the entire rams with elevated levels associated with regressed activity. Prolactin secretion in the ovariectomized ewes was significantly (P less than 0.05) related to the phase of ovarian development with high levels associated with acyclic activity. It is concluded that LH secretion and pituitary responsiveness to exogenous GnRH were not modified by the artificial light regime. However, the changing light pattern was physiologically 'perceived' by the castrate animals as indicted by a concomitant variation in plasma prolactin concentrations.

The proposed use of melatonin in controlled sheep breeding.

The regulation by melatonin of hypothalamic-pituitary events in the ewe to advance seasonal oestrous activity, with no undesirable effects upon fertility, and its induction of those seasonal responses associated with short days indicates an essential role for melatonin in controlled-breeding programs in major sheep-producing countries. The development of suitable controlled-release systems to provide a choice of practical methods of melatonin delivery under field conditions is discussed as also are geographical and breed factors in controlled breeding with melatonin.

The response of rams and ewes of three breeds to artificial photoperiod.

Rams and ewes of the Romney Marsh (N = 6), Dorset Horn (N = 8) and Australian Merino (N = 8) breeds were subjected to 4 successive periods of alternating 6 h light/18 h dark ('short' days) and 18 h light/6 h dark ('long' days) preceded by 16 weeks of 12 h light/12 h dark. The initial period was of 32 weeks (16 weeks 'short' days; 16 weeks 'long' days) and the next 3 were of 24 weeks (12 weeks 'short' days; 12 weeks 'long' days). Rams of all breeds showed a cyclic pattern of growth and regression of testes associated with plasma testosterone concentration, influenced by the change in light regimen 15-19 weeks previously. Sexual behaviour was also cyclic but lagged by some 6-7 weeks. The changes were greatest in the Romneys and least in the Merinos in which a higher degree of sexual activity was evident even when the testes were regressed (P less than 0.001). This was the major breed difference. All ewes of the Romney and Dorset breeds showed marked seasonality related to the imposed light regimen, whereas only 1 of the 4 Merinos did so. The mean peak of ovarian activity in the former 2 breeds coincided with that of maximum sexual activity of rams housed with them; that is, some 6 weeks after maximum scrotal volume. The rams and ewes were subjected to serial blood sampling episodes for plasma LH and testosterone and tested for plasma LH release following GnRH administration. There was little variation between breeds in LH concentration. Testosterone concentration varied greatly in the ram, highest levels associated with the developed phase of the testes and with maximum LH pulse frequency. The LH response to GnRH changed with respect to the state of the gonads. Maximal responses were observed in the developing phase of testicular growth although this variation was greater in the Romney and Dorsets than in the Merinos (P less than 0.001). In the ewes, maximal responses were seen in the follicular phase (P less than 0.001), with no difference between the luteal and acyclic phases. There were no breed differences. Plasma pooled from the serial blood sampling episodes was assayed for prolactin.(ABSTRACT TRUNCATED AT 400 WORDS)

Plasma prolactin concentrations in pinealectomized ewes receiving melatonin treatment and in pineal intact ewes maintained under a non-24-hour photoperiod.

We have investigated the profiles of prolactin secretion in relation to onset of breeding activity in Suffolk-Cross ewes with artificially modified melatonin rhythms. With treatments commencing in midsummer, groups of ewes were 1) subjected to a 8L:16D photoperiod, 2) maintained under a "short" (6L:16D) photoperiod repeated over a 22-h cycle that induces an elevation in plasma melatonin that does not endure for the entire dark phase, 3) pinealectomized (pnx) to abolish plasma melatonin levels, and 4) pinealectomized and treated with a melatonin implant (subcutaneous) to provide a constant (no 24-h rhythm) elevation in plasma melatonin. The onset of breeding activity was significantly advanced in both the 8L:16D and pnx/implant groups compared to the 6L:16D and untreated pnx ewes. The two latter groups displayed a normal timing in seasonal breeding activity. Low and high plasma prolactin levels corresponded with short and long photoperiods during both the 24 and 22-h cycles. There was no clearcut "seasonal" rhythm in plasma prolactin in either of the pnx groups. A clear differentiation was seen between reproductive response and prolactin response, particularly in the case of ewes monitored on 22-h cycles of short photoperiod.

How does melatonin control seasonal reproductive cycles?

The pineal gland is essential for the perception of photoperiod change in many species. Information about photoperiod length is conveyed through pineal secretion of the methoxyindole melatonin. Melatonin, suitably administered in physiological quantities is equipotent with artificial photoperiod in the induction of photoperiodic responses. Most experimental work suggests that it is the duration of high night time melatonin secretion (positively correlated with the length of the natural or artificial dark phase) which conveys the photoperiodic signal. Continuous release implants induce short day effects in ewes, entirely comparable to daily feeding of melatonin or short photoperiod. A minimum duration of secretion rather than a specific duration is therefore probably critical to short day effects. There appears to be a seasonal variation in sensitivity to short day melatonin effects (induction of early oestrus) which can be shifted to an earlier time of year following one oestrus advance the previous year. Short duration melatonin is read as a long day even secreted with 22 hour periodicity, suggesting a lack of circadian variation in sensitivity to melatonin.

Pineal and ovarian response to 22- and 24-h days in the ewe.

Melatonin secretion in ewes was entrained by 22-h light-dark cycles whether of long (16L:6D) or short (6L:16D) photoperiod. In photoperiods of 6L:16D, a phase-delay of melatonin secretion was evident, leading to a dark-phase duration shorter than that found in 8L:16D. Early onset of estrus was induced in anestrous ewes kept in 8L:16D, but not 6L:16D, from 22 July compared to controls in natural light. In photoperiods of 16L:6D, the melatonin profile corresponded precisely to the dark phase. Early offset of estrus was induced in estrous ewes kept in both 18L:6D and 16L:6D from 18 December compared to controls in natural light. Thus, when the duration of melatonin secretion was appropriate to the long photoperiod (16L:6D), but with a constantly changing phase position, a long-day reproductive response was found. Activity-rest cycles were not entrained by 16L:6D; thus the synchronization of melatonin and activity-rest cycles does not appear to be essential for the induction of a long-day reproductive response. These results support the hypothesis that the duration, not the circadian-phase position, of melatonin is critical to the induction of photoperiodic effects.

Intraruminal soluble glass boluses containing melatonin can induce early onset of ovarian activity in ewes.

Soluble phosphate glass boluses (preparations A, B, and C) have been developed to release melatonin into the reticulo-rumen of ewes at relatively different (fast, medium or slow) rates. A fourth type (preparation D) containing no melatonin was used as a sham control. Four groups of seasonally anoestrous Suffolk-cross ewes were respectively dosed with preparations A, B, C or D on 4 July. Plasma samples were collected twice weekly for melatonin and progesterone assay. In Groups A, B and C, elevated daytime plasma concentrations of melatonin could be detected for about 5 weeks after bolus administration. However, the pattern of hormone release was variable between groups, with Group C animals maintaining higher plasma melatonin concentrations for a longer period. The control animals had undetectable daytime melatonin levels. The onset of cyclic ovarian activity in the animals treated with the 'slow' release bolus (Group C) was significantly (P less than 0.05) advanced compared to the control group. The 'fast' and 'medium' release treatments (Groups A and B) did not significantly alter the onset of ovarian activity. The results indicate the potential of a novel and convenient method of melatonin delivery for induction of early breeding activity in ewes.

Metabolism and pharmacokinetics of melatonin in the ewe.

The pineal hormone melatonin has been used to advance the onset of the breeding season in sheep and thus produce lambs earlier in the year. If this reproductive manipulation is to be used commercially, some knowledge of the route of metabolism and identity of possible metabolites is necessary. A major metabolite of melatonin in rodents and man is 6-hydroxymelatonin sulphate (acetyl-methoxytryptamine-6-sulphate [aMT6S]). No significant amounts of this metabolite could be found in the plasma of untreated ewes. After subcutaneous implantation of melatonin for 5 months, plasma levels of aMT6S were also insignificant. On the other hand, both a single oral dose of melatonin (3 mg) and daily oral dosing gave rise to circulating levels of aMT6S in the range of 150 to 1,500 pg/ml for at least 18 h. The profiles seen after 180 days treatment were similar to those seen after a single dose, indicating that this route of melatonin metabolism is not induced by chronic administration. Intravenous injection of melatonin (200 or 20 micrograms) gave rise to detectable levels of aMT6S in the plasma. These results indicate that the quantitative aspects of melatonin metabolism differ according to the route of administration.

A comparison of the efficiency of melatonin treatments in advancing oestrus in ewes.

Early oestrous cycles were induced in adult, maiden, 18-month-old Suffolk-cross ewes, maintained from birth in natural photoperiod by the following treatments applied from mid-June: subcutaneous implantation of melatonin (1 g) in Silastic packets, daily, oral, melatonin administration (3 mg/ewe) at 15:30 h, an artificial photoperiod of 8L:16D (lights on 07:30 h). Ovarian cycles began 5-10 weeks before those of control ewes maintained in a natural photoperiod. In contrast, the onset of ovarian cycles in ewes given s.c. implants of melatonin (1 g) in April, and a further group in May, was highly variable, and not significantly different from that of the control ewes. Plasma melatonin profiles in sheep with implants showed a night-time rise super-imposed on a constant level, which was itself within the physiological night-time range. Implant-derived melatonin declined with time but remained at or above physiological night-time levels for at least 3 1/2 months. These results indicate that melatonin implants in June, but not in April or May, advance onset of oestrus in the non-lactating, adult ewe. The effects of melatonin implants in June on onset of ovarian cycles were indistinguishable from those of melatonin feeding or artificial short photoperiod initiated at this time of year.

Suppression of nocturnal plasma melatonin and 6-sulphatoxymelatonin by bright and dim light in man.

Previous studies have shown that bright light (2500 lux) suppresses nocturnal secretion of melatonin, while dim light (500 lux) has little or no effect. We have studied the effect of varying intensities of light on 5 normal male volunteers (age 18-28). The experiment was divided into 3 parts which took place at weekly intervals. Subjects remained under artificial light (fluorescent strip 150-250 lux) between 2000 h-2300 h, they then retired to bed in darkness. On each occasion, between 0030 h and 0100 h, the subjects were required to get up and were treated with light of different intensities; (a) less than 1 lux, (b) 300 lux and (c) 2500 lux respectively. Subjects returned to bed in darkness until 0700 h. Blood was sampled hourly from 2000 h-1000 h with additional samples at 2330 h, 0015 h, 0030 h, 0045 h, 0115 h and 0130 h. Plasma melatonin and 6-sulphatoxymelatonin (aMT6s), the major melatonin metabolite, were measured by radioimmunoassay. Dim (300 lux) and bright (2500 lux) light, both significantly suppressed melatonin levels compared to less than 1 lux (P less than 0.05 and P less than 0.01 respectively) at the following time points 0100 h, 0115 h and 0130 h. One subject did not show suppression with 300 lux. There was also a significant suppression of aMT6s levels, compared to less than 1 lux, after both 300 lux and 2500 lux at 0115 h (P less than 0.05, P less than 0.01), 0130 h (P less than 0.01, P less than 0.01) and 0200 h (P less than 0.01, P less than 0.001) respectively.(ABSTRACT TRUNCATED AT 250 WORDS)