Plasma Melatonin Concentrations Research Articles

Effects of Melatonin on the Defense to Acute Hypoxia in Newborn Lambs.

Neonatal lambs, as other neonates, have physiologically a very low plasma melatonin concentration throughout 24 h. Previously, we found that melatonin given to neonates daily for 5 days decreased heart weight and changed plasma cortisol and gene expression in the adrenal and heart. Whether these changes could compromise the responses to life challenges is unknown. Therefore, firstly, we studied acute effects of melatonin on the defense mechanisms to acute hypoxia in the neonate. Eleven lambs, 2 weeks old, were instrumented and subjected to an episode of acute isocapnic hypoxia, consisting of four 30 min periods: normoxia (room air), normoxia after an i.v. bolus of melatonin (0.27 mg kg−1, n = 6) or vehicle (ethanol 1:10 NaCl 0.9%, n = 5), hypoxia (PaO2: 30 ± 2 mmHg), and recovery (room air). Mean pulmonary and systemic blood pressures, heart rate, and cardiac output were measured, and systemic and pulmonary vascular resistance and stroke volume were calculated. Blood samples were taken every 30 min to measure plasma norepinephrine, cortisol, glucose, triglycerides, and redox markers (8-isoprostane and FRAP). Melatonin blunted the increase of pulmonary vascular resistance triggered by hypoxia, markedly exacerbated the heart rate response, decreased heart stroke volume, and lessened the magnitude of the increase of plasmatic norepinephrine and cortisol levels induced by hypoxia. No changes were observed in pulmonary blood pressure, systemic blood pressures and resistance, cardiac output, glucose, triglyceride plasma concentrations, or redox markers. Melatonin had no effect on cardiovascular, endocrine, or metabolic variables, under normoxia. Secondly, we examined whether acute melatonin administration under normoxia could have an effect in gene expression on the adrenal, lung, and heart. Lambs received a bolus of vehicle or melatonin and were euthanized 30 min later to collect tissues. We found that melatonin affected expression of the immediate early genes egr1 in adrenal, ctgf in lung, and nr3c1, the glucocorticoid receptor, in adrenal and heart. We speculate that these early gene responses may contribute to the observed alterations of the newborn defense mechanisms to hypoxia. This could be particularly important since the use of melatonin is proposed for several diseases in the neonatal period in humans.

Effect of melatonin on monochromatic light-induced changes in clock gene circadian expression in the chick liver

Light is the most prominent zeitgeber of the circadian system, which contains central and peripheral oscillators. Our previous studies found that light wavelength could influence the rhythms of melatonin synthesis and clock gene expression in the central oscillator of chicks. However, the effect of monochromatic light on the peripheral oscillator and the role of melatonin have yet to be clarified. In this study, 216 newly hatched chicks were divided into three groups (intact, sham operation and pinealectomy) and were raised under white (WL), red (RL), green (GL) or blue (BL) light for 14 days. Their plasma and livers were sampled at 6 time points with 4-h intervals. Plasma melatonin concentration and liver clock gene expression (cClock, cBmal1, cBmal2, cCry1, cCry2, cPer2, cPer3) were measured for circadian rhythm analysis. In intact and sham operation chicks under WL, all liver clock genes showed circadian expression along with oscillations in plasma melatonin. However, positive clock genes peaked at subjective night along with melatonin, while negative clock genes peaked at subjective day or the shifting time of day-night. Chick exposure to monochromatic light led to an unaltered circadian rhythmicity in plasma melatonin and liver clock genes; however, their rhythmic parameters were notably influenced. Compared to WL, GL enhanced the mesor and amplitude of melatonin and all kinds of clock genes, whereas RL had the opposite effect. Pinealectomy significantly decreased expression of liver clock genes, which was consistent with the reduction in plasma melatonin concentration, especially for the GL group, and resulted in the expression of liver clock genes showing low-mesor and low-amplitude oscillations as well as no statistically significant differences among the monochromatic light groups. Thus, we speculated that melatonin plays a key role in the effects of light wavelength on clock gene rhythm in the chick liver.

Dim light at night attenuates circadian rhythms in the cardiovascular system and suppresses melatonin in rats

AimsCardiovascular parameters exhibit significant 24-h variability, which is coordinated by the suprachiasmatic nucleus (SCN), and light/dark cycles control SCN activity. We aimed to study the effects of light at night (ALAN; 1–2 lx) on cardiovascular system control in normotensive rats. Main methodsHeart rate (HR) and blood pressure (BP) were measured by telemetry during five weeks of ALAN exposure. From beat-to-beat telemetry data, we evaluated spontaneous baroreflex sensitivity (sBRS). After 2 (A2) and 5 (A5) weeks of ALAN, plasma melatonin concentrations and the response of BP and HR to norepinephrine administration were measured. The expression of endothelial nitric oxide synthase (eNOS) and endothelin-1 was determined in the aorta. Spontaneous exploratory behaviour was evaluated in an open-field test. Key findingsALAN significantly suppressed the 24-h variability in the HR, BP, and sBRS after A2, although the parameters were partially restored after A5. The daily variability in the BP response to norepinephrine was reduced after A2 and restored after A5. ALAN increased the BP response to norepinephrine compared to the control after A5. Increased eNOS expression was found in arteries after A2 but not A5. Endothelin-1 expression was not affected by ALAN. Plasma melatonin levels were suppressed after A2 and A5. Spontaneous exploratory behaviour was reduced. SignificanceALAN decreased plasma melatonin and the 24-h variability in the haemodynamic parameters and increased the BP response to norepinephrine. A low intensity ALAN can suppress circadian control of the cardiovascular system with negative consequences on the anticipation of a load.

Melatonin Levels in Preterm and Term Infants and Their Mothers.

The prevention of perinatal brain damage following preterm birth remains a public health priority. Melatonin has been shown to be a promising neuroprotectant in neonatal preclinical models of brain damage, but few studies have investigated melatonin secretion in newborns. We hypothesized that melatonin circulating levels would be lower in preterm compared to term infants. We conducted a prospective, longitudinal, multicenter study to assess melatonin, and 6-sulfatoxy-melatonin (aMT6s) concentrations, measured by radioimmunoassay. Among 209 neonates recruited, 110 were born before 34 gestational weeks (GW) and 99 born after 34 GW. Plasma melatonin concentrations, measured at birth and on Day 3 were below detectable levels (≤7 pg/mL) in 78% and 81%, respectively, of infants born before 34 GW compared to 57% and 34%, respectively, of infants born after 34 GW. The distribution of plasma melatonin concentrations was found to be correlated with gestational age at both time-points (p < 0.001). Median urine aMT6s concentrations were significantly lower in infants born before 34 GW, both on Day 1 (230 ng/L vs. 533 ng/L, p < 0.0001) and on Day 3 (197 ng/L vs. 359 ng/L, p < 0.0001). In conclusion, melatonin secretion appears very low in preterm infants, providing the rationale for testing supplemental melatonin as a neuroprotectant in clinical trials.

Melatonin-improved buffalo semen quality during nonbreeding season under tropical condition

The role of melatonin in protecting spermatozoa from different kinds of injury has been widely reported. The present study aimed to test whether treatment of buffalo bulls with melatonin could ameliorate sperm function during nonbreeding season under tropical condition. Ten Murrah buffalo bulls were randomly allocated into control and treated groups of equal numbers of bulls to study the effect of melatonin on semen characteristics, seminal plasma constituents, blood plasma hormonal levels, and antioxidant enzyme activities during nonbreeding season. Treated bulls were implanted with melatonin (18 mg/50 kg of body weight) for a period of 2 mo. During this period, semen was collected twice a week, and blood samples were collected weekly to determine plasma concentration of melatonin and LH and activities of antioxidant enzymes. During nonbreeding season, melatonin implantation improved semen characteristics by increasing (P < 0.05) percentages of sperm with forward motility, viability, total motile sperm, and rapid motility, average path, curvilinear, and straight-line velocity and amplitude of lateral head displacement and decreasing (P < 0.05) percentages of abnormal sperm and linearity index as compared to the control group. Furthermore, melatonin implantation increased (P < 0.05) seminal plasma concentrations of total protein, albumin, and cholesterol and decreased (P < 0.05) seminal plasma aspartate aminotransferase activity. In addition, melatonin-implanted bulls exhibited an increase (P < 0.05) in red blood cells superoxide dismutase activity compared to untreated bulls. In conclusion, melatonin implantation successfully improved semen quality of buffalo bulls during nonbreeding season under tropical condition.

PK-PD based optimal dose and time for orally administered supra-pharmacological dose of melatonin to prevent radiation induced mortality in mice

AimsThe study reports preclinical pharmacokinetics (PK) and correlation with pharmacological effect at suprapharmacological dose of orally administered melatonin along with time and dose optimization, which have been lacking in earlier reports of radioprotection using melatonin. MethodsPK of melatonin in C57BL/6 mice was evaluated after dose of 250 mg/kg using HPLC. Tissue distribution study was conducted in vital organs following oral administration. Plasma total antioxidant capacity (TAC) was determined by ABTS+ radical assay and was correlated to plasma concentrations of melatonin. Using the outcomes of PK and Pharmacodynamics (PD), survival study was conducted for optimization of ‘drug radiation gap period’ (DRGP). Optimal oral dose for radioprotection was determined using survival as an end point. Key findingsPK analysis of melatonin revealed Tmax at 5 min with closely spaced another distinct concentration peak at 20 min. Plasma TAC of melatonin showed similar peaks at 5 min and 45 min, with the highest TAC at 45 min. Survival following a lethal (9 Gy) radiation dose was 20% and 40% after 5 and 45 min of melatonin administration, respectively. DRGP for melatonin was thus 45 min, while optimal oral dose ranged from 125 to 250 mg/kg. PK parameters at 250 mg/kg dose were qualitatively similar to low dose of melatonin, thus preventing chances of unexpected toxicity. SignificanceSurvival enhancement at 45 min suggested as probable interval required as ‘DRGP’. The optimum oral therapeutic window appears large with no substantial toxicity. The outcomes will be useful in development of radioprotectors as well as other therapeutic applications.

Pineal gland calcification under hypoxic conditions

The pineal gland (glandula pinealis) is neuroendocrine gland located at the epithalamus of the brain secreting melatonin. The aim of this study was to explore effects of prenatal hypoxia in rats at the age of 33 weeks on the occurrence of pineal gland calcification. Distribution and chemical composition of calcerous material by light, scanning and transmission electron microscopy was investigated. Melatonin concentrations in blood plasma by direct radioimmunoassay were measured. Rats were exposed to prenatal hypoxia for 12 h at day 20 of development and second group to prenatal hypoxia for 2x8 h at days 19 and 20 of development. Vacuoles of intracellular edema in the pineal samples after 12 h hypoxia were found. Their size ranges up to 30 µm. Some of them were filled with the flocculent and fibrous material. Samples of pineal glands after 2 x 8 h hypoxia revealed the pericellular edema of pinealocytes. The amount of calcium rich particles in 2 x 8 h hypoxia group was lower than in 12 h hypoxia group. Plasma melatonin levels did not differ between control and both hypoxia groups. We concluded that calcification is a process induced by osteoblasts and osteocytes with melatonin as a promotor and it is favored under hypoxic conditions.

Changes in melatonin concentrations in seminal plasma are not correlated with testosterone or antioxidant enzyme activity when rams are located in areas with an equatorial photoperiod

In temperate climates, photoperiod and melatonin regulate ram reproduction, modulating hormonal secretions, sperm quality, and seminal plasma composition. Information on the effect of an equatorial photoperiod (12L:12D) on ram reproduction, however, is scarce, and no data on hormonal concentrations and antioxidant enzyme activity in seminal plasma have been reported. Thus, the variation was investigated of melatonin and its relationship with testosterone and antioxidant enzyme activity in the seminal plasma of three sheep breeds in Colombia, when there was a consistent photoperiod during two dry and two rainy seasons per year. Semen was collected once a week from 12 mature rams (four of each breed: Colombian Creole, Hampshire, and Romney Marsh). Seminal plasma was obtained by centrifugation. The concentration of melatonin and testosterone were quantified along with the enzymatic activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase (CAT). Correlation analyses between melatonin and testosterone concentrations or enzymatic activity were also performed. Melatonin concentration was affected by season (P < 0.05) but not breed, with lesser concentrations in the first rainy season. Testosterone concentration, however, was affected by breed and season, with greater concentrations (P < 0.01) in the Hampshire and Romney Marsh rams during the second dry season. Regarding antioxidant enzyme activity, there was only seasonal variation in GPx activity (P < 0.05). When correlation analyses were used for data assessments, there was a negative correlation between melatonin and testosterone concentrations in Hampshire rams. In conclusion, melatonin concentrations in seminal plasma of rams that were located in an area with an equatorial photoperiod was affected by the climatological season but there was no positive correlation with testosterone concentration or antioxidant enzyme activity.

Milk yield, lactation parameters and prolactin secretion characteristics in sheep treated with melatonin implants during pregnancy and lactation in long-day conditions

In a temperate climate zone, sheep lambing during long days (summer) produce less milk and have shorter lactation than those naturally lambing during short days (winter). We investigated whether artificially induced and prolonged increase in melatonin concentration in sheep lambed during long days would improve milk yield and lactation parameters through changes in pituitary prolactin (PRL) secretion. In addition, the relative mRNA levels of PRL and PRL-receptor (PRL-R) were determined in the mammary gland. Sheep (n = 36) were randomly divided into three groups: lambed in January (short day – SD treatment group), lambed in June (long day – LD treatment group), and lambed in June and treated with the subcutaneous melatonin implants (in May and then in August, LDM treatment group). After lamb weaning, milk yield, the number of milking days and the total length of lactation (days) were monitored individually. Plasma melatonin and PRL concentrations were determined in samples collected from 18 sheep (6 of each group): 6 weeks before parturition, day after weaning and then during lactation, every 28 days. A mammary gland biopsy was performed in these sheep 3–4 days before parturition. Milk yield was higher in the SD group, than in the LD (P < 0.05) and LDM (P < 0.001) groups. Both the number of milking days and total length of lactation were higher in the SD (P < 0.001) and LDM (P < 0.05) groups than in the LD group. The PRL and melatonin secretion profiles varied in all groups, reflecting the characteristics of the endogenous circannual rhythm. An additional increase in plasma PRL concentration occurred in LDM sheep, compared to LD sheep (P < 0.01). Statistical differences were found neither in the relative levels of PRL mRNA nor PRL-R mRNA in the mammary gland between the SD and LD groups, however both transcripts contents decreased (P < 0.05) in the LDM group. In conclusions: 1) alterations in milk yield and lactational parameters in sheep are affected by changes in the secretory PRL profile during the established lactation; 2) PRL refractoriness to melatonin develops in lactating sheep with extended melatonin treatment, which increases only a number of milking days, with no positive effect on milk yield; 3) The sheep mammary gland is qualitatively at the same level of preparation, in both short and long days, for the synthesis and perception of PRL during the periparturient period; 4) Artificially elevated plasma melatonin concentration before parturition causes a decrease in abundance of both PRL and PRL-R transcripts in the mammary gland.

Spontaneously Diabetic Torii (SDT) Fatty Rat, a Novel Animal Model of Type 2 Diabetes Mellitus, Shows Blunted Circadian Rhythms and Melatonin Secretion

In patients with diabetes mellitus (DM), impairments of circadian rhythms, including the sleep–wake cycle, blood pressure, and plasma melatonin concentrations, are frequently observed. Animal models of DM are also reported to show aberrant circadian rhythms. However, the changes in the circadian rhythms of plasma soluble substances, including melatonin, in diabetic animals are controversial. In the present study, we investigated the circadian rhythms of spontaneous locomotor activity, metabolic parameters (plasma glucose, triglyceride, and total cholesterol), and plasma melatonin concentrations in Spontaneously Diabetic Torii (SDT) fatty rats, a novel animal model of type 2 DM. Although SDT fatty rats exhibited low locomotor activity in the dark phase, no phase shifts were observed. The circadian variations of plasma metabolic parameters were more apparent in the SDT fatty rats compared with control Sprague–Dawley (SD) rats. The circadian rhythms of plasma melatonin concentrations were significantly impaired in SDT fatty rats. To get an insight into the mechanism underlying the impaired melatonin secretion in SDT fatty rats, the expression of arylalkylamine N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt) mRNA, which encode the rate-limiting enzymes for melatonin synthesis, was investigated in the pineal gland. There were no significant differences in Aanat and Asmt expression between the control SD and SDT fatty rats. These results suggest that SDT fatty rats show impaired circadian rhythms and dysregulated melatonin secretion.

Preparation, characterization and in vitro evaluation of melatonin-loaded porous starch for enhanced bioavailability

The present work aimed to apply porous starch (PS) as carrier to improve the oral bioavailability of poorly soluble drug melatonin (MLT). The drug loading and encapsulation efficiency of MLT-loaded porous starch (MPS) were optimized. The characteristics of MPS were analyzed using scanning electron microscopy, Brunauer–Emmett–Teller surface area, Fourier–transform infrared spectroscopy, x-ray diffraction, differential scanning calorimetry and thermo gravimetric analysis. Most MLT transformed into their amorphous form in the PS pores. MPS showed higher MLT solubility and cumulative release rate compared with raw MLT in SGF and SIF. MPS exhibited a higher inhibition to DCFH–DA-oxidized peroxyl radicals at a lower EC50 than that of the raw MLT. Furthermore, the plasma concentrations of MLT and MPS reached a Cmax of 134.26 and 291.77 ng/mL at 15 and 20 min, respectively. The AUC0–360min of the formulated MPS-treated group was approximately 2.34-fold higher than that of raw MLT.

Interplay between the APOE Genotype and Possible Plasma Biomarkers in Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with a complex pathogenesis and a common occurrence of comorbid diseases such as depression. It is accepted that the presence of the ε4 allele of the gene that encodes apolipoprotein E (APOE) is the strongest genetic risk factor for the development of sporadic AD. Melatonin, cortisol, homocysteine, and prolactin are presumed to be risk factors or biomarkers for stress- and age-related disorders. The interplay between the APOE genotype and plasma biomarkers was examined in patients with AD presenting with or without depression to contribute to understanding the interdependence of various molecular mechanisms in the pathophysiology of AD. The APOE genotype and morning plasma melatonin, cortisol, homocysteine, and prolactin concentrations were measured in 85 patients with AD and 44 elderly controls. A significant association between AD and the allele (ε4) or genotype (ε3/ε4 or ε4/ε4) frequencies of APOE was confirmed. Plasma homocysteine and cortisol levels were significantly increased in patients with AD compared to those in controls, independent of the presence of comorbid depressive symptoms or the severity of dementia. Significantly lower plasma melatonin concentration was found in patients with AD but not in controls, who were noncarriers of the APOE ε4 allele, regardless of the presence of depression or the severity of dementia in AD. Our findings indicate the existence of a little-known specific APOE-mediated mechanism that increases the plasma melatonin level in a subgroup of patients with AD who are carriers of the APOE ε4 allele.

P101. The influence of sex and age on melatonin suppression by warm-white light (K 2883 K)

Introduction In the past, the suppression of the pineal hormone melatonin by light has been investigated in several studies, frequently using small samples and only male subjects. Whereas the nocturnal melatonin production is thought to decrease with age, our knowledge about the extent of the light-induced melatonin suppression depending on sex and age is only limited. Our study addressed this issue in more details. Methods Subjects for this study were pre-screened for the onset of melatonin production in the evening hours. In total, 94 healthy subjects between 18 and 73 years were enrolled (42 men/52 women). All of them had a regular sleep-wake schedule and were morning or intermediate types according to the Ostberg-Horne-Ostberg morningness-eveningness questionnaire (scores: 41–73). They completed 4 evening laboratory sessions (19:00–23:00 h) predominantly in a weekly interval. The light protocol for each session consisted of a 2-h dim light ( Results After 2 h of dim-light condition all enrolled study participants showed an increase in melatonin production versus baseline of different magnitude. One hour of warm-white light at 500 lx (0.264 W/m2) caused very variable suppression of melatonin which did not exceed about 64% in both sexes. The average melatonin suppression over all subjects was 30.2% with sex related differences in the frequency of strong suppression, being higher in men. The inter-individual variability in melatonin suppression by warm-weight light was not related to the age of study participants. We also proved the impact of physical activity. Men with low physical activity showed relatively small plasma melatonin concentrations after 2 h dim-light condition, but a high sensitivity to the warm-white light applicated. Conclusions The obvious individual variability in the sensitivity of melatonin production for artificial light in the evening with possible sex but not clear and simple age related differences could be a critical factor in our understanding of light effects. Levels of physical activity in the leisure time could be a masking factor which should be adequately controlled in similar studies.

CHANGES OF THE SLEEP, HORMONAL BALANCE AND PROFILE OF CYTOKINES IN PREGNANT WOMEN WITH PLACENTAL INSUFFICIENCY

Objective – to establish the correlation between the placental insufficiency, sleep disorders and pro- and anti-inflammatory part of the immune system by questioning, studying of the levels of melatonin, placental hormones and pro- and anti-inflammatory cytokines in pregnant women with placental insufficiency.Materials and methods. 80 pregnant women with placental insufficiency, manifesting as intrauterine fetal growth restriction (IUGR) of II-III degree in the 3rd pregnancy trimester (study group) and 30 women with normal clinical flow of pregnancy (control group) were questioned. 46 patients from study group and 20 patients from control group women were examined further: the studying of the blood plasma concentrations of melatonin, placental hormones (placental lactogen, free estriol), pro-inflammatory cytokines, such as tumor necrotizing factor-α (TNF-α), interleukin-1-β (IL-1-β), interleukin-6 (IL-6), and anti-inflammatory cytokines, such as interleukin-4 (IL-4) and interleukin-10 (IL-10), was conducted.Results and the discussion. Questioning showed that pregnant women with placental insufficiency (PI) in 86% cases experience the sleep disorders starting from pregnancy term 12-22 weeks (healthy pregnant women – mostly after 30 weeks, 57% cases), more commonly wake up 2 or more times per night (71% of positive answers, in control group – 23%), and 3 or more times per week (78% of positive answers, in control group – 17%). It also has been established that concentration of melatonin significantly decreases, if the pregnancy is complicated by intrauterine fetal growth restriction (study group – 126,87±14,87 pg/ml, control group – 231,25±21,56 pg/ml, p&lt;0,001). The levels of pro-inflammatory cytokines in the study group were significantly higher, comparing with the control group (TNF-α: study group – 10,05±1,35 pg/ml, control group – 5,60±1,50 pg/ml, p&lt;0,05; IL-1-β: study group – 14,67±2,13 pg/ml, control group – 3,96±0,92 pg/ml, p&lt;0,001; IL-6: study group – 6,91±0,99 pg/ml, control group – 2,69±0,99 pg/ml, p&lt;0,05). The same is true about anti-inflammatory cytokines (IL-4: study group – 5,97±0,50 pg/ml, control group – 3,74±0,62 pg/ml, p&lt;0,05; IL-10: study group – 11,40±1,50 pg/ml, control group – 4,70±3,20 pg/ml, p&lt;0,001). We did not find significant difference in hormonal concentrations between groups.Conclusions. Sleep disorders in pregnant women with placental insufficiency appear earlier and seem to be more expressed then in healthy pregnant women. The blood level of melatonin significantly decreases in case of placental insufficiency, manifested as intrauterine fetal growth restriction. The strengthening of the pro-inflammatory immunity, shown as the increasing of the levels of TNF-α, IL-1-β and IL-6, is also present in case of IUGR. The rising of the plasma concentration of the anti-inflammatory cytokines, such as IL-4 and IL-10, in our opinion, can be explained by activation of the mechanisms of compensation, which decrease the risk of premature labor.

Use of anti-phosphotyrosine and PDK1 monoclonal antibodies enables observation of the effects of media on bull sperm second messenger signaling during capacitation

In temperate climates, photoperiod and melatonin regulate ram reproduction, modulating hormonal secretions, sperm quality, and seminal plasma composition. Information on the effect of an equatorial photoperiod (12L:12D) on ram reproduction, however, is scarce, and no data on hormonal concentrations and antioxidant enzyme activity in seminal plasma have been reported. Thus, the variation was investigated of melatonin and its relationship with testosterone and antioxidant enzyme activity in the seminal plasma of three sheep breeds in Colombia, when there was a consistent photoperiod during two dry and two rainy seasons per year. Semen was collected once a week from 12 mature rams (four of each breed: Colombian Creole, Hampshire, and Romney Marsh). Seminal plasma was obtained by centrifugation. The concentration of melatonin and testosterone were quantified along with the enzymatic activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase (CAT). Correlation analyses between melatonin and testosterone concentrations or enzymatic activity were also performed. Melatonin concentration was affected by season (P < 0.05) but not breed, with lesser concentrations in the first rainy season. Testosterone concentration, however, was affected by breed and season, with greater concentrations (P < 0.01) in the Hampshire and Romney Marsh rams during the second dry season. Regarding antioxidant enzyme activity, there was only seasonal variation in GPx activity (P < 0.05). When correlation analyses were used for data assessments, there was a negative correlation between melatonin and testosterone concentrations in Hampshire rams. In conclusion, melatonin concentrations in seminal plasma of rams that were located in an area with an equatorial photoperiod was affected by the climatological season but there was no positive correlation with testosterone concentration or antioxidant enzyme activity.

Characterization of interleukin-6 in the caprine testis

In temperate climates, photoperiod and melatonin regulate ram reproduction, modulating hormonal secretions, sperm quality, and seminal plasma composition. Information on the effect of an equatorial photoperiod (12L:12D) on ram reproduction, however, is scarce, and no data on hormonal concentrations and antioxidant enzyme activity in seminal plasma have been reported. Thus, the variation was investigated of melatonin and its relationship with testosterone and antioxidant enzyme activity in the seminal plasma of three sheep breeds in Colombia, when there was a consistent photoperiod during two dry and two rainy seasons per year. Semen was collected once a week from 12 mature rams (four of each breed: Colombian Creole, Hampshire, and Romney Marsh). Seminal plasma was obtained by centrifugation. The concentration of melatonin and testosterone were quantified along with the enzymatic activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase (CAT). Correlation analyses between melatonin and testosterone concentrations or enzymatic activity were also performed. Melatonin concentration was affected by season (P < 0.05) but not breed, with lesser concentrations in the first rainy season. Testosterone concentration, however, was affected by breed and season, with greater concentrations (P < 0.01) in the Hampshire and Romney Marsh rams during the second dry season. Regarding antioxidant enzyme activity, there was only seasonal variation in GPx activity (P < 0.05). When correlation analyses were used for data assessments, there was a negative correlation between melatonin and testosterone concentrations in Hampshire rams. In conclusion, melatonin concentrations in seminal plasma of rams that were located in an area with an equatorial photoperiod was affected by the climatological season but there was no positive correlation with testosterone concentration or antioxidant enzyme activity.

The effects of leptin on plasma concentrations of prolactin, growth hormone, and melatonin vary depending on the stage of pregnancy in sheep.

The effects of hyperleptinemia and leptin resistance during gestation are unclear. Leptin, an important neuroendocrine regulator, has anorexic effects, but its interactions with other metabolic hormones during pregnancy are unclear. We examined potential roles of leptin in regulating prolactin (PRL), GH, and melatonin plasma concentrations during pregnancy in Polish Longwool ewes. Twelve estrus-synchronized ewes carrying twins after mating were randomly assigned to receive i.v. injections of saline or recombinant ovine leptin (2.5 or 5.0 µg/kg BW). Blood samples were collected (15-min intervals over 4 h) immediately before the first injection at dusk and kept under red light. Treatments were repeated at 2-wk intervals, starting before mating and continuing from days 30 to 135 of gestation. Concentrations of plasma PRL, GH, and melatonin were determined using a validated RIA. The effects of leptin on hormone plasma concentrations varied depending on pregnancy stage and leptin dose. PRL plasma concentrations were affected at most stages of pregnancy and before gestation. In non-, very early- (day 30), and late- (day 120 and 135) pregnant ewes, exogenous leptin stimulated PRL (P < 0.001) plasma concentrations, while during the second month of gestation, it decreased PRL concentrations (P < 0.01). Leptin affected GH plasma concentrations (P < 0.05) only during the first 2 mo of pregnancy, with no effects during the second part of gestation or before pregnancy. In early-pregnant ewes (day 30 and 45), leptin decreased melatonin plasma concentrations (P < 0.05), but at day 60, leptin stimulated melatonin plasma concentrations at low (P < 0.01) and high doses (P < 0.05), with no effects in ewes after 105 d of gestation. These data indicate specific pregnancy-induced endocrine adaptations to changes in energy homeostasis, supporting the hypothesis that leptin affects PRL, GH, and melatonin release during gestation.

Effects of melatonin administration on seminal plasma metabolites and sperm fertilization competence during the non-reproductive season in ram

The purpose of this study was to investigate the effects of ram melatonin treatment on the sperm quality and metabolite composition of the seminal plasma in the non-breeding season. Four mature rams were treated with 54 mg melatonin in March subcutaneous implants and four untreated rams served as control. At 0, 30, 90 and 120 days semen samples were collected and sperm, separated from seminal plasma, was evaluated for its capacity to fertilize and produce embryos in vitro. Seminal plasma metabolites were extracted and analyzed by capillary electrophoresis/mass spectroscopy. In the resulting electropherograms, the area corresponding to selected metabolites was extracted and quantified. Ram melatonin treatment affected the in vitro fertilization competence of sperm. Blastocyst output increased until 90 days after treatment (27.20 ± 7.35 vs 54.7 ± 4.4% at 0 and 90 days respectively; p < 0.05) while the untreated group did not show statistical differences.In treated rams, the concentration of melatonin in seminal plasma increased from 3.34 ± 1.70 at day 0–9.65 ± 2.89 AU (Arbitrary Units) after 90 days, then decreased to reach the level of the untreated ram after 120 days (p < 0.05). During 90 days after melatonin treatment, an increase (p < 0.05) in seminal plasma concentrations of glutamic acid (6.28 ± 1.53 vs 14.93 ± 1.53 AU at 0 and 90 days respectively), glutamine (16.89 ± 4.65 vs 54.51 ± 4.65 AU), carnitine (22.97 ± 9.81 vs 104.30 ± 9.81 AU), acetyl-carnitine (48.15 ± 17.32 vs 217.69 ± 17.32 AU), choline (1.82 ± 1.55 vs 14.16 ± 1.55 AU) and arginine (1.31 ± 1.08 vs 14.25 ± 1.08 AU) was detected. Tyrosine concentration increased during 30 days from melatonin treatment (12.79 ± 3.93 vs 27.08 ± 3.04 AU) but at 90 days its levels were similar to the untreated group. In conclusion, melatonin treatment during the non-breeding season improves the concentration of several metabolites in seminal plasma and sperm fertilization competence in Sarda breed ram.

Effect of Melatonin Implants during the Non-Breeding Season on the Onset of Ovarian Activity and the Plasma Prolactin in Dromedary Camel.

To examine a possible control of reproductive seasonality by melatonin, continual-release subcutaneous melatonin implants were inserted 4.5 months before the natural breeding season (October–April) into female camels (Melatonin-treated group). The animals were exposed to an artificial long photoperiod (16L:8D) for 41 days prior to implant placement to facilitate receptivity to the short-day signal that is expected with melatonin implants. The treated and control groups (untreated females) were maintained separately under outdoor natural conditions. Ovarian follicular development was monitored in both groups by transrectal ultrasonography and by plasma estradiol-17β concentrations performed weekly for 8 weeks and then for 14 weeks following implant insertion. Plasma prolactin concentrations were determined at 45 and 15 days before and 0, 14, 28, 56, and 98 days after implant insertion. Plasma melatonin concentration was determined to validate response to the artificial long photoperiod and to verify the pattern of release from the implants. Results showed that the artificial long photoperiod induced a melatonin secretion peak of significantly (P < 0.05) shorter duration (about 2.5 h). Melatonin release from the implants resulted in higher circulating plasma melatonin levels during daytime and nighttime which persisted for more than 12 weeks following implants insertion. Treatment with melatonin implants advanced the onset of follicular growth activity by 3.5 months compared to untreated animals. Plasma estradiol-17β increased gradually from the second week after the beginning of treatment to reach significantly (P < 0.01) higher concentrations (39.2 ± 6.2 to 46.4 ± 4.5 pg/ml) between the third and the fifth week post insertion of melatonin implants. Treatment with melatonin implants also induced a moderate, but significant (P < 0.05) suppressive effect on plasma prolactin concentration on the 28th day. These results demonstrate that photoperiod appears to be involved in dromedary reproductive seasonality. Melatonin implants may be a useful tool to manipulate seasonality and to improve reproductive performance in this species. Administration of subcutaneous melatonin implants during the transition period to the breeding season following an artificial signal of long photoperiod have the potential to advance the breeding season in camels by about 2.5 months.

Melatonin-Mediated Development of Ovine Cumulus Cells, Perhaps by Regulation of DNA Methylation.

Cumulus cells of pre-pubertal domestic animals are dysfunctional, perhaps due to age-specific epigenetic events. This study was designed to determine effects of melatonin treatment of donors on methylation modification of pre-pubertal cumulus cells. Cumulus cells from germinal vesicle stage cumulus oocyte complexes (COCs) were collected from eighteen lambs which were randomly divided into control group (C) and melatonin group given an 18 mg melatonin implant subcutaneous (M). Compared to the C group, the M group had higher concentrations of melatonin in plasma and follicular fluid (p < 0.05), greater superovulation, a higher proportion of fully expanded COCs, and a lower proportion of apoptotic cumulus cells (p < 0.05). Real-time PCR results showed that melatonin up-regulated expression of genes MT1, Bcl2, DNMT1, DNMT3a and DNMT3b, but down-regulated expression of genes p53, Caspase 3 and Bax (p < 0.05). Furthermore, melatonin increased FI of FITC (global methylation level) on cumulus cells (p < 0.05). To understand the regulation mechanism, the DNMTs promoter methylation sequence were analyzed. Compared to the C group, although there was less methylation at two CpG sites of DNMT1 (p < 0.05) and higher methylation at two CpG sites of DNMT3a (p < 0.05), there were no significant differences in methylation of the detected DNMT1 and DNMT3a promoter regions. However, there were lower methylation levels at five CpG sites of DNMT3b, which decreased methylation of detected DNMT3b promoter region on M group (p < 0.05). In conclusion, alterations of methylation regulated by melatonin may mediate development of cumulus cells in lambs.