12D Cycle Research Articles

Circadian Rhythm Alteration of the Core Clock Genes and the Lipid Metabolism Genes Induced by High-Fat Diet (HFD) in the Liver Tissue of the Chinese Soft-Shelled Turtle (Trionyx sinensis).

Physiology disorders of the liver, as it is an important tissue in lipid metabolism, can cause fatty liver disease. The mechanism might be regulated by 17 circadian clock genes and 18 fat metabolism genes, together with a high-fat diet (HFD). Due to their rich nutritional and medicinal value, Chinese soft-shelled turtles (Trionyx sinensis) are very popular among the Chinese people. In the study, we aimed to investigate the influence of an HFD on the daily expression of both the core clock genes and the lipid metabolism genes in the liver tissue of the turtles. The two diets were formulated with 7.98% lipid (the CON group) and 13.86% lipid (the HFD group) to feed 180 juvenile turtles, which were randomly divided into two groups with three replicates per group and 30 turtles in each replicate for six weeks, and the diet experiment was administrated with a photophase regimen of a 24 h light/dark (12L:12D) cycle. At the end of the experiment, the liver tissue samples were collected from nine turtles per group every 3 h (zeitgeber time: ZT 0, 3, 6, 9, 12, 15, 18, 21 and 24) for 24 h to investigate the daily expression and correlation analysis of these genes. The results showed that 11 core clock genes [i.e., circadian locomotor output cycles kaput (Clock), brain and muscle arnt-like protein 1 and 2 (Bmal1/2), timeless (Tim), cryptochrome 1 (Cry2), period2 (Per2), nuclear factor IL-3 gene (Nfil3), nuclear receptor subfamily 1, treatment D, member 1 and 2 (Nr1d1/2) and retinoic acid related orphan receptor α/β/γ β and γ (Rorβ/γ)] exhibited circadian oscillation, but 6 genes did not, including neuronal PAS domain protein 2 (Npas2), Per1, Cry1, basic helix-loop-helix family, member E40 (Bhlhe40), Rorα and D-binding protein (Dbp), and 16 lipid metabolism genes including fatty acid synthase (Fas), diacylglycerol acyltransferase 1 (Dgat1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), Low-density lipoprotein receptor-related protein 1-like (Ldlr1), Lipin 1 (Lipin1), Carnitine palmitoyltransferase 1A (Cpt1a), Peroxisome proliferator activation receptor α, β and γ (Pparα/β/γ), Sirtuin 1 (Sirt1), Apoa (Apoa1), Apolipoprotein B (Apob), Pyruvate Dehydrogenase kinase 4 (Pdk4), Acyl-CoA synthase long-chain1 (Acsl1), Liver X receptors α (Lxrα) and Retinoid X receptor, α (Rxra) also demonstrated circadian oscillations, but 2 genes did not, Scd and Acaca, in the liver tissues of the CON group. However, in the HFD group, the circadian rhythms' expressional patterns were disrupted for the eight core clock genes, Clock, Cry2, Per2, Nfil3, Nr1d1/2 and Rorβ/γ, and the peak expression of Bmal1/2 and Tim showed delayed or advanced phases. Furthermore, four genes (Cry1, Per1, Dbp and Rorα) displayed no diurnal rhythm in the CON group; instead, significant circadian rhythms appeared in the HFD group. Meanwhile, the HFD disrupted the circadian rhythm expressions of seven fat metabolism genes (Fas, Cpt1a, Sirt1, Apoa1, Apob, Pdk4 and Acsl1). Meanwhile, the other nine genes in the HFD group also showed advanced or delayed expression peaks compared to the CON group. Most importantly of all, there were remarkably positive or negative correlations between the core clock genes and the lipid metabolism genes, and their correlation relationships were altered by the HFD. To sum up, circadian rhythm alterations of the core clock genes and the lipid metabolism genes were induced by the high-fat diet (HFD) in the liver tissues of T. sinensis. This result provides experimental and theoretical data for the mass breeding and production of T. sinensis in our country.

Altered circadian rhythms effects on anxiety, locomotor activity, serotonin and nitric oxide levels in the prefrontal cortex of juvenile and adult rats

AbstractBackgroundDementia and Alzheimer’s disease (AD) can be exacerbated by irregular sleep‐wake cycles, and age may be a risk factor. This study compared behavioral responses such as anxiety, motor coordination, and locomotion to altered circadian rhythms and serotonin, nitric oxide (NO), and malondialdehyde (MDA) levels in the prefrontal cortex of juvenile and adult rats.MethodA total of thirty female Wistar rats were grouped into three (n = 5) in the following juvenile (30‐45 days old) and adult (90‐120 days old) groups: (A) Control (12L:12D cycle),(B) 24‐hour LED light exposure(12L:12L cycle) and (C) 24‐hour dark cycle (12D:12D cycle). Experimental rats were kept in the stated cycle conditions using natural lighting, LED lighting, and a dark room, respectively, for 60 days. Data were analyzed using a two‐way ANOVA (GraphPad Prism 9), and statistical significance was measured at p<0.05.ResultFindings from the study revealed progressive weight loss in the sleep‐deprived juvenile (12L:12L) rats compared to adults (p<0.05) in the same and other groups. Also, anxiety‐like indices such as longer duration of time in the dark area of the light‐dark box, avoidance of open arm entries on the elevated plus maze, reduced line crossing activity in the open field box, etc. were markedly higher in the juvenile rats from the 12L:12L and 12D:12D groups respectively. When juvenile and adult 12L:12L and 12D:12D cycle rats data were compared to controls, severe depletion of serotonin (p<0.0001) and nitric oxide (p<0.01) levels were observed, while age‐dependent MDA levels were revealed across all groups.ConclusionA bi‐directional relationship was observed between age and circadian disruption effects with more severe outcomes in juveniles compared to adults and this hold interesting implications for the study of the biological clock mechanisms underlying anxiety, mood and sleep disorders in living systems.

Monochromatic Green Light Stimulation during Incubation Alters Hepatic Glucose Metabolism That Improves Embryonic Development in Yangzhou Goose Eggs

The influence of monochromatic green light stimulation on hatching performance and embryo development has been studied in chickens, but not geese. The liver has crucial functions in the regulation of energy metabolism during embryogenesis, but its involvement in green light transduction is still unidentified. We aimed to determine the influence of monochromatic green light on Yangzhou goose hatching performance and embryo development. We also investigated the metabolomics and transcriptomic responses of the embryonic liver to green light to determine the underlying molecular mechanisms. Eggs were incubated under either 12 h of monochromatic green light/dark (12 L:12D) cycles or 24 h of darkness (0G:24D). Green light promoted embryonic development and hatching performance, also affected the expression of myogenic regulatory factors associated with muscle development. It also shortened hatching time and elevated plasma levels of growth hormone and insulin-like growth factor-1. Metabolomics and transcriptomic results revealed differentially expressed genes and metabolites with enhanced gluconeogenesis/glycolysis and increased plasma glucose and pyruvate levels under green light. Hence, the growth-promoting effect possibly through regulating energy metabolism in the liver and myogenic regulatory factors in muscle. Our findings provide important and novel insights into the mechanisms underlying the beneficial effects of green light on goose embryos.

Agarose hydrogel composite supports microgreen cultivation with enhanced porosity and continuous water supply under terrestrial and microgravitational conditions

Hydrogels are attractive soilless media for plant cultivation with strong water and nutrient retention. However, pristine hydrogels contain mostly ultra-micro pores and lack air-filled porosity for root zone aeration. Herein we report a porous hydrogel composite comprising an agarose network and porous growing mix particle (GMP) fillers. The agarose backbone allowed the composite to sustain a 12-d growth cycle for red cabbage microgreens without the need for watering or crew interaction. Moreover, the GMP induced greater total pore volume and increased the prevalence of pores >30 μm by 8-fold. Further investigation suggested that the nutrients from GMP accounted for a 54 % increase in microgreen yield over pristine hydrogel, while the porous structure introduced by GMP improved the yield by another 44 %. Increased air-filled porosity accelerated the water transport and loss of hydrogel but maintained favorable water potential levels for plant extraction. Finally, the hydrogel composite supported microgreen growth satisfyingly under simulated microgravity despite some morphological changes. Results of this study reveal a novel growth substrate that is lightweight, convenient, and water-efficient, while effectively sustaining plant growth for multiple applications including indoor farming and space farming.

Embryonic Development of Avian Pineal Secretory Activity-A Lesson from the Goose Pineal Organs in Superfusion Culture.

The embryonic ontogeny of pineal secretory activity in birds has been investigated almost exclusively in chickens. This study aimed to characterize this process in domestic geese. The pineal organs of embryos aged 18–28 days were incubated in superfusion culture under different light conditions for 4–5 days and treated with norepinephrine (NE). Melatonin (MLT) was measured by radioimmunoassay and other indoles by HPLC with fluorescence detection. Additionally, pineal organs were collected from embryos at 14–28 days of age and used to measure catecholamines by HPLC with electrochemical detection. MLT secretion increased with embryo age, most intensively between the 22nd and 24th days of life. The daily changes in MLT secretion under the 12 L:12D cycle occurred on the first day of culture, starting from an embryonic age of 24 days. MLT secretion was controlled by the light-dark cycle in all age groups studied. However, exposure to light during the scotophase did not alter the secretion of MLT. The endogenous oscillator expressed its activity in regulating MLT secretion in the pineal organs of embryos aged 24 days and older but could not generate a rhythm after one cycle. The rhythm of 5-hydroxytryptophan release during the first day of culture was found in the pineal organs of all embryos, while the rhythmic release of N-acetylserotonin and 5-methoxyindole acetic acid started at the age of 24 days. The proportion of released indoles changed with embryo age. NE caused a decrease in MLT secretion and provoked an increase in serotonin release. Incubation of the pineal organs induced the development of MLT secretory machinery and its diurnal rhythmicity. The pineal content of catecholamines increased prominently at the end of embryonic development.

Expression patterns of iron regulatory proteins after intense light exposure in a cone-dominated retina.

Iron is implicated in ocular diseases such as in age-related macular degeneration. Light is also considered as a pathological factor in this disease. Earlier, two studies reported the influence of constant light environment on the pattern of expressions of iron-handling proteins. Here, we aimed to see the influence of light in 12-h light-12-h dark (12L:12D) cycles on the expression of iron-handling proteins in chick retina. Chicks were exposed to 400 lx (control) and 5000 lx (experimental) light at 12L:12D cycles and sacrificed at variable timepoints. Retinal ferrous ion (Fe2+) level, ultrastructural changes, lipid peroxidation level, immunolocalization and expression patterns of iron-handling proteins were analysed after light exposure. Both total Fe2+ level (p = 0.0004) and lipid peroxidation (p = 0.002) significantly increased at 12-, 48- and 168-h timepoint (for Fe2+) and 48- and 168-h timepoint (for lipid peroxidation), and there were degenerative retinal changes after 168 h of light exposure. Intense light exposure led to an increase in the levels of transferrin and transferrin receptor-1 (at 168-h) and ferroportin-1, whereas the levels of ferritins, hephaestin, (at 24-, 48- and 168-h timepoint) and ceruloplasmin (at 168-h timepoint) were decreased. These changes in iron-handling proteins after light exposure are likely due to a disturbance in the iron storage pool evident from decreased ferritin levels, which would result in increased intracellular Fe2+ levels. To counteract this, Fe2+ is released into the extracellular space, an observation supported by increased expression of ferroportin-1. Ceruloplasmin was able to convert Fe2+ into Fe3+ until 48 h of light exposure, but its decreased expression with time (at 168-h timepoint) resulted in increased extracellular Fe2+ that might have caused oxidative stress and retinal cell damage.

Low-input cultivation of camelina (<em>Camelina sativa</em> (L.) Crantz) in a Mediterranean semi-arid environment

The cultivation of oil crops for biofuel production has often been accused of not being environmentally sustainable due to the high inputs needed. To explore the effect of reduced input on productive and qualitative traits of camelina (Camelina sativa (L.) Crantz), a trial was carried out over a two-year period. This study analysed two different levels of input: a low input treatment (shallow non-inversion tillage and low fertilisation rate) and a high input treatment (deeper tillage and high fertilisation rate). Camelina was positively, even though to a limited extent, affected by high input treatment as highlighted by the increase in seed yield (from 1.8 to 2.0 t ha–1), crop residues (from 4.8 to 5.2 t ha–1), seed protein content (from 26.5 to 28.9%), seed oil content (from 41.5 to 43.4%) and oil yield (from 0.75 to 0.88 t ha–1). So, from a sustainable point of view, we must consider negligible the effect of high input and satisfactory the performances of camelina in the low input regime. Low input management resulted in satisfactory yields in terms of both quantity and quality, results which were not very different from high input, indicating promising potential for conservation agriculture practices in camelina in a semi-arid environment.
 Highlights - Camelina showed high adaptability to conservation agriculture practices. - Seed yield of 1.9 t ha–1 was obtained under reduced tillage and low fertilisation rate. - High inputs (fertilisation and tillage) determined a 12-d longer crop cycle. - α-linolenic, erucic and eicosenoic acids were affected by input levels.

Effect of constant light on the morphology of the retina in domestic chick (Gallus g. domesticus): A scanning electron microscope study

Light induces variable levels of damage to the retina and often, death of the photoreceptor cells. It is reported that chick retina, havingabundant melanin in the retinal pigment epithelium, undergoes no or minimal damage from exposure to light. However, the morphology of cone and rod photoreceptor cells after continuous exposure of light has not been studied in detail. The present study investigated the detailed changes of photoreceptor cells, by scanning electron microscopy (SEM).Domestic chicks (Gallus g. domesticus) from post-hatch day (PH) 1 to 7 were initially reared at 700 lux at 12 h light (L): 12 h dark (D). From PH 7 to 18, they were exposed to 12L: 12D (control) and 24L: 0D (constant light) cycles at 700 lux. At PH18, chicks were sacrificed, their eyes enucleated and preserved in Karnovsky’s fixative. Theretina was then separated from the adherent choroid, mounted on photoreceptor side up and processed for SEM. There was significant damage to rod and cone outer segmentsfrom both dorsal and ventral part of the retina and decrease in the abundance of cells of inner nuclear layer in constant light, compared to that found in 12L: 12D cycles. It is likely that constant light affects the retinal cells in the vertical pathway, which could alter the photo transduction process.

Changes in the Inner Retinal Cells after Intense and Constant Light Exposure in Sprague‐Dawley Rats

Light insult causes photoreceptor death. Few studies reported that continuous exposure to light affects horizontal, Müller and ganglion cells. We aimed to see the effect of constant light exposure on bipolar and amacrine cells. Adult Sprague-Dawley rats were exposed to 300 or 3000 lux for 7days in 12-h light: 12-h dark cycles (12L:12D). The latter group was then exposed to 24L:0D for 48h to induce significant damage. The same animals were reverted to 300lux and reared for 15days in 12L:12D cycles. They were sacrificed on different days to find the degree of retinal recovery, if any, from light injury. Besides photoreceptor death, continuous light for 48h resulted in downregulation of parvalbumin in amacrine cells and recoverin in cone bipolar cells (CBC). Rod bipolar cells (RBC) maintained an unaltered pattern of PKC-α expression. Upon reversal, there were increased expressions of parvalbumin in amacrine cells and recoverin in CBC, while RBC showed an increasing trend of PKC-α expression. The data show that damage in bipolar and amacrine cells after exposure to intense, continuous light can be ameliorated upon reversal to normal LD cycles to which the animals were initially acclimated to.

Diurnal Rhythm of Plasma Melatonin Concentration in the Domestic Turkey and Its Regulation by Light and Endogenous Oscillators.

Simple SummaryEnvironmental light regulates a wide range of phenomena in almost all organisms on Earth. Daily and seasonal changes in the photoperiod duration are the most important factors controlling the secretion of melatonin (MLT), a pineal hormone that affects many physiological processes in birds. The results of previous studies on the effect of MLT on the productivity and health of poultry have been promising. However, there are very few studies on the daily profiles of plasma MLT concentrations in domestic birds; therefore, we decided to examine plasma MLT levels in 10-week-old domestic turkeys exposed to different light conditions. The results demonstrated that plasma MLT concentration in turkeys kept under a 12 h light: 12 h dark cycle showed a prominent diurnal rhythm. Night-time light exposure caused a rapid decrease in plasma MLT concentrations. The housing of turkeys in continuous dim red light revealed endogenously generated diurnal rhythm of MLT secretion. The rhythm of the plasma MLT level in a reversed cycle of 12 h dark: 12 h light adapted quickly to the new lighting condition. The aim of this study was to characterize the diurnal rhythm of plasma melatonin (MLT) concentration and its regulation by light and endogenous oscillators in 10-week-old domestic turkeys. Three experiments were performed to examine (i) the course of daily changes in plasma MLT concentration in turkeys kept under a 12 h light: 12 h dark (12L:12D) cycle; (ii) the influence of night-time light exposure lasting 0.5, 1, 2, or 3 h on the plasma MLT level; and (iii) the occurrence of circadian fluctuations in plasma MLT levels in birds kept under continuous dim red light and the ability of turkeys to adapt their pineal secretory activity to a reversed light-dark cycle (12D:12L). The plasma MLT concentration was measured with a direct radioimmunoassay. The plasma MLT concentration in turkeys kept under a 12L:12D cycle changed significantly in a daily rhythm. It was low during the photophase and increased stepwise after the onset of darkness to achieve the maximal level in the middle of the scotophase. Next, it decreased during the second half of the night. The difference between the lowest level of MLT and the highest level was approximately 18-fold. The exposure of turkeys to light during the scotophase caused a rapid, large decrease in plasma MLT concentration. The plasma MLT concentration decreased approximately 3- and 10-fold after 0.5 and 1 h of light exposure, respectively, and reached the day-time level after 2 h of exposure. In turkeys kept under continuous darkness, the plasma MLT level was approximately 2.5-fold higher at 02:00 h than at 14:00 h. In birds kept under 12D:12L, the plasma MLT level was significantly higher at 14:00 h than at 02:00 h. The results showed that plasma MLT concentrations in 10-week-old turkeys have a prominent diurnal rhythm, which is endogenously generated and strongly influenced by environmental light.

Embryonic Ontogeny of 5-Hydroxyindoles and 5-Methoxyindoles Synthesis Pathways in the Goose Pineal Organ.

The aim of this study was to characterize the embryonic ontogeny of 5-hydroxyindoles and 5-methoxyindoles synthesis pathways in the goose pineal organ. The study was performed on embryos aged 14–28 days, which have been incubated under a 12L:12D cycle. The pineal organs were collected for measurements of indole content by HPLC every 6 h on embryonic day (ED) 14, ED 16, ED 18 and ED 22 or every 2 h on ED 24, ED 26 and ED 28. The level of tryptophan showed no significant changes during development and no day-night variations. The content of 5-hydroxytryptophan increased between ED 14 and ED 26. It was significantly higher during scotophase than during photophase starting from ED 14. The serotonin content was low during the early stages of development (ED 14–ED 18) and prominently increased from ED 20. The serotonin levels also showed day-night differences; however, they were less conspicuous than those of 5-hydroxytryptophan. The changes in the level of 5-hydroxyindole acetic acid were similar to those of serotonin. 5-Hydroxytryptophol was measurable from ED 18. Levels of N-acetylserotonin, which were detectable for the first time on ED 16, prominently increased between ED 22 and ED 28 and showed significant day–night differences from ED 20. Melatonin was detectable from ED 18. Like N-acetylserotonin, its content increased rapidly between ED 22 and ED 28, and from ED 20 showed diurnal variations. 5-Methoxyindole acetic acid and 5-methoxytryptophol occurred at measurable levels from ED 18 and ED 26, respectively. The obtained results showed that embryonic development of indole metabolism in the goose pineal organ starts with the beginning of serotonin synthesis. The processes of serotonin acetylation and 5-hydroxyindoles methylation were turned on later. Diurnal rhythmicity develops very early in the embryonic pineal organ of the goose when the eggs are incubated under a 12 h light: 12 h dark schedule. Two processes are responsible for generation of the diurnal rhythms of 5-hydroxyindoles and 5-methoxyindoles: (i) hydroxylation of tryptophan and (ii) acetylation of serotonin.

The effect of different wavelengths of light during incubation on the development of rhythmic pineal melatonin biosynthesis in chick embryos

Rhythmic pineal melatonin biosynthesis develops in chick embryos incubated under a light (L)-dark (D) cycle of polychromatic white light. The spectral sensitivity of the embryonic pineal gland is not known and was investigated in this study. Broiler breeder eggs (Ross 308, n=450) were incubated under white, red, green or blue light under the 12L : 12D cycle. Melatonin was measured in extracts of pineal glands by radioimmunoassay. The daily rhythm of pineal melatonin levels in 20-day-old chick embryos was confirmed during the final stages of embryonic life under all four wavelengths of light with expected higher concentrations during dark- than light-times. The highest pineal melatonin levels were determined in chick embryos incubated under red and white light and lower levels under green light. The incubation under blue light resulted in the lowest melatonin biosynthesis. Pineal melatonin concentrations increased substantially on post-hatching day two compared with pre-hatching levels and we did not find differences between birds incubated and kept in either white or green light. Our results demonstrate a selective sensitivity of the chick embryo pineal gland to different wavelengths of light. Rhythmic melatonin production is suggested as a possible mechanism, which transfers information about the quality of ambient light to the developing avian embryo.

The effect of high fat diet on daily rhythm of the core clock genes and muscle functional genes in the skeletal muscle of Chinese soft-shelled turtle (Trionyx sinensis).

In the present study, we sought to investigate the influence of high fat diet on the core clock genes and the muscle functional genes daily expression in the skeletal muscle of Chinese soft-shelled turtle. The turtles were fed by two diets including a control fat diet (the CON treatment, 7.98% lipid) and a high fat diet (the HFD treatment, 13.86% lipid) for six weeks and administrated by the photophase regimen of 24h light/dark (12L:12D) cycle. After the feeding trial experiment, we measured the daily expression levels of 17 core clock genes (Clock, Bmal1/2, NPAS2, Tim, Cry1/2, Per1/2, DBP, AANAT, NIFL3, BHLHE40, NR1D2, RORA, RORB, RORC) and 12 muscle functional genes (FBXO32, MBNL1, MSTN, Myf5, Myf6, MyoD, MyoG, MyoM1, PPARa, PDK4, Trim63, UCP3) in the skeletal muscle of the two treatments. The results showed that except for Bmal1, NPAS2, Per2 and RORB, the expression of the other 13 core clock genes exhibited circadian oscillation in the CON treatment. Among the 12 muscle functional genes, MBNL1, PDK4 and MyoM1 did not exhibit circadian oscillation in the CON treatment. In the HFD treatment, the circadian rhythms expressional patterns of the 8 core clock genes (Clock, Bmal2, Cry2, Per1, DBP, NFIL3, BHLHE40 and RORA) and 6 muscle functional genes (MSTN, Myf5, MyoD, MyoG, PPARa and Trim63) were disrupted. In addition, compared with the CON treatment, the circadian expression of the 5 core clock genes (Tim, Cry1, AANAT, NR1D2, RORC) and the 3 muscle functional genes (FBXO32, Myf6, UCP3) showed the advanced or delayed expression peaks in the HFD treatment. In CON treatment, the circadian expression of the MyoG, MyoD, Myf6, FBXO32 and PPARa showed positive or negative correlation with the transcription pattern of Clock, Bmal2, Cry1/2, Per1/2. However, only the FBXO32 and Myf6 presented positive or negative correlation with the circadian expression of Cry1, RORB, AANAT and Tim in HFD treatment. In summary, these results demonstrate that the disruption of the circadian rhythm of the core clock genes and muscle functional genes in the skeletal muscle is closely associated with feeding high fat diet to Chinese soft-shelled turtle.

Genome-Wide Scleral Micro- and Messenger-RNA Regulation During Myopia Development in the Mouse.

PurposeMicroRNA (miRNAs) have been previously implicated in scleral remodeling in normal eye growth. They have the potential to be therapeutic targets for prevention/retardation of exaggerated eye growth in myopia by modulating scleral matrix remodeling. To explore this potential, genome-wide miRNA and messenger RNA (mRNA) scleral profiles in myopic and control eyes from mice were studied.MethodsC57BL/6J mice (n = 7; P28) reared under a 12L:12D cycle were form-deprived (FD) unilaterally for 2 weeks. Refractive error and axial length changes were measured using photorefraction and 1310-nm spectral-domain optical coherence tomography, respectively. Scleral RNA samples from FD and fellow control eyes were processed for microarray assay. Statistical analyses were performed using National Institute of Aging array analysis tool; group comparisons were made using ANOVA, and gene ontologies were identified using software available on the Web. Findings were confirmed using quantitative PCR in a separate group of mice (n = 7).ResultsForm-deprived eyes showed myopic shifts in refractive error (−2.02 ± 0.47 D; P < 0.01). Comparison of the scleral RNA profiles of test eyes with those of control eyes revealed 54 differentially expressed miRNAs and 261 mRNAs fold-change >1.25 (maximum fold change = 1.63 and 2.7 for miRNAs and mRNAs, respectively) (P < 0.05; minimum, P = 0.0001). Significant ontologies showing gene over-representation (P < 0.05) included intermediate filament organization, scaffold protein binding, detection of stimuli, calcium ion, G protein, and phototransduction. Significant differential expression of Let-7a and miR-16-2, and Smok4a, Prph2, and Gnat1 were confirmed.ConclusionsScleral mi- and mRNAs showed differential expression linked to myopia, supporting the involvement of miRNAs in eye growth regulation. The observed general trend of relatively small fold-changes suggests a tightly controlled, regulatory mechanism for scleral gene expression.

Effects of acute stress on aggression and the cortisol response in the African sharptooth catfish Clarias gariepinus: differences between day and night.

African sharptooth catfish Clarias gariepinus were housed under continuous dim light (1 lx) or 12L:12D (350-0 lx) cycles. The number of skin lesions, as indicator of aggressive acts, and plasma cortisol levels, as indicator of stress-axis activity, were measured at baseline as well as following a stressor (given in the light or dark phase). Results showed that (1) baseline plasma cortisol levels were not different between photoperiods, (2) the number of baseline skin lesions was highest for C. gariepinus housed under continuous dim light, (3) stressor-induced peak levels of plasma cortisol were highest in the light phase and (4) the number of skin lesions following a stressor was highest in the dark phase. The higher number of stressor-related skin lesions in the dark (active) phase suggests increased stressor-induced aggression while in the active phase. In addition, the data suggest that housing under continuous dim light does not result in higher stress-axis activity, as measured by baseline levels of cortisol, but does result in more stressor-induced aggression, as measured by the higher number of skin lesions. The latter may be related to the fact that the continuous dim light photoperiod has twice the number of dark-phase (active) hours in which stressor-induced aggression is stronger compared to the 12L:12D photoperiod, which has a light phase in which stressor-induced aggression is lower.

Diurnal profiles of melatonin synthesis-related indoles, catecholamines and their metabolites in the duck pineal organ.

This study characterizes the diurnal profiles of ten melatonin synthesis-related indoles, the quantitative relations between these compounds, and daily variations in the contents of catecholamines and their metabolites in the domestic duck pineal organ. Fourteen-week-old birds, which were reared under a 12L:12D cycle, were killed at two-hour intervals. The indole contents were measured using HPLC with fluorescence detection, whereas the levels of catecholamines and their metabolites were measured using HPLC with electrochemical detection. All indole contents, except for tryptophan, showed significant diurnal variations. The 5-hydroxytryptophan level was approximately two-fold higher during the scotophase than during the photophase. The serotonin content increased during the first half of the photophase, remained elevated for approximately 10 h and then rapidly decreased in the middle of the scotophase. N-acetylserotonin showed the most prominent changes, with a more than 15-fold increase at night. The melatonin cycle demonstrated only an approximately 5-fold difference between the peak and nadir. The 5-methoxytryptamine content was markedly elevated during the scotophase. The 5-hydroxyindole acetic acid, 5-hydroxytryptophol, 5-methoxyindole acetic acid and 5-methoxytryptophol profiles were analogous to the serotonin rhythm. The norepinephrine and dopamine contents showed no significant changes. The DOPA, DOPAC and homovanillic acid levels were higher during the scotophase than during the photophase. Vanillylmandelic acid showed the opposite rhythm, with an elevated level during the daytime.

Ameliorative effects of melatonin administration and photoperiods on diurnal fluctuations in cloacal temperature of Marshall broiler chickens during the hot dry season.

Experiments were performed with the aim of determining the effect of melatonin administration on diurnal fluctuations in cloacal temperature (CT) of Marshall broiler chickens during the hot dry season. Birds in group I (12L:12D cycle) were raised under natural photoperiod of 12-h light and 12-h darkness, without melatonin supplementation, while those in group II (LL) were kept under 24-h continuous lighting, without melatonin administration. Broiler chickens in group III (LL + melatonin) were raised under 24-h continuous lighting, with melatonin supplementation at 0.5 mg/kg per os. The cloacal temperatures of 15 labeled broiler chickens from each group were measured at 6:00, 13:00, and 19:00 h, 7 days apart, from days 14-42. Temperature-humidity index was highest at day 14 of the study, with the value of 36.72 ± 0.82 °C but lowest at day 28 with the value of 30.91 ± 0.80 °C (P < 0.0001). The overall mean hourly cloacal temperature value of 41.51 ± 0.03 °C obtained in the 12L:12D cycle birds was significantly higher (P < 0.001) than the value of 41.16 ± 0.03 °C recorded in the melatonin-treated group but lower than that of 41.65 ± 0.03 °C obtained in the LL birds. Mortality due to hyperthermia commenced at day 28 in both 12L:12D cycle and LL broiler chickens but was delayed till day 42 in LL + MEL broiler chickens. In conclusion, melatonin administration alleviated the deleterious effects of heat stress on broiler chickens by maintaining their cloacal temperature at relatively low values.

Cloning of circadian rhythmic pathway genes and perturbation of oscillation patterns in endocrine disrupting chemicals (EDCs)-exposed mangrove killifish Kryptolebias marmoratus

To investigate the effect of endocrine disrupting chemicals (EDCs) on the circadian rhythm pathway, we cloned clock and circadian rhythmic pathway-associated genes (e.g. Per2, Cry1, Cry2, and BMAL1) in the self-fertilizing mangrove killifish Kryptolebias marmoratus. The promoter region of Km-clock had 1 aryl hydrocarbon receptor element (AhRE, GTGCGTGACA) and 8 estrogen receptor (ER) half-sites, indicating that the AhRE and ER half sites would likely be associated with regulation of clock protein activity during EDCs-induced cellular stress. The Km-clock protein domains (bHLH, PAS1, PAS2) were highly conserved in five additional fish species (zebrafish, Japanese medaka, Southern platyfish, Nile tilapia, and spotted green pufferfish), suggesting that the fish clock protein may play an important role in controlling endogenous circadian rhythms. The promoter regions of Km-BMAL1, -Cry1, -Cry2, and -Per2 were found to contain several xenobiotic response elements (XREs), indicating that EDCs may be able to alter the expression of these genes. To analyze the endogenous circadian rhythm in K. marmoratus, we measured expression of Km-clock and other circadian rhythmic genes (e.g. Per2, Cry1, Cry2, and BMAL1) in different tissues, and found ubiquitous expression, although there were different patterns of transcript amplification during different developmental stages. In an estrogen (E2)-exposed group, Km-clock expression was down-regulated, however, a hydroxytamoxifen (TMX, nonsteroid estrogen antagonist)-exposed group showed an upregulated pattern of Km-clock expression, suggesting that the expression of Km-clock is closely associated with exposure to EDCs. In response to the exposure of bisphenol A (BPA) and 4-tert-octyphenol (OP), Km-clock expression was down-regulated in the pituitary/brain, muscle, and skin in both gender types (hermaphrodite and secondary male). In juvenile K. marmoratus liver tissue, expression of Km-clock and other circadian rhythmic pathway-associated genes showed a regular oscillation pattern over a period of approximately 24h during a 12L:12D cycle. However, the circadian rhythm of BPA-exposed juvenile K. marmoratus liver tissue was perturbed over a 12L:12D period. This study will aid in our understanding of how EDCs perturb endogenous circadian rhythms, particularly in BPA-exposed fish liver tissue.

The effects of the duration and onset of light stimulation during incubation on the behavior, plasma melatonin levels, and productivity of broiler chickens1

Light stimulation during incubation can affect the behavior, health, and performance of poultry posthatch. However, there has been relatively little work systematically assessing the pattern of light stimulation needed to produce these effects or the mechanism underlying them. We conducted 2 experiments to assess the effects of duration and onset of light exposure during incubation on Cobb 500 broiler chickens. In the first, eggs (n = 1,404) were incubated under photoperiods of either 0 h of light and 24 h of darkness (0 L:24 D), 1 h of light and 23 h of darkness (1 L:23 D), 6 h of light and 18 h of darkness (6L:18D), or 12 h of light and 12 h of darkness (12L:12D). In the second, eggs (n = 1,008) were incubated in either complete darkness or under 12L:12D, which was applied either for the entire incubation period or with light onset beginning at either 7 or 14 d of incubation. Broilers were then housed in floor pens under a 12L:12D cycle posthatch. Measurements included performance outcomes, plasma melatonin, general behavioral activity assessed using passive infrared detection, and feeding activity assessed using automated continuous monitoring of feed intake at wk 5 of age. There were no treatment differences in hatchability, mortality, growth, feed consumption, feed conversion ratio, overall feeding behavior activity, or general behavioral activity over a 24-h period in either experiment. However, broilers incubated under 12L:12D fed more (P < 0.05) than the 0 L:24 D broilers during the first 3 h after the lights came on in Exp. 1 and during the first hour after the lights came on in Exp. 2. In Exp. 1, general activity levels measured using passive infrared detection at night also differed (P = 0.05), with 0 L:24 D more active than 12L:12D. There was a treatment difference between the 0 L:12 D and 12 L:12 D in their plasma melatonin rhythms during d 19 of incubation, but this difference had disappeared when broilers were sampled at wk 5 posthatch. The results of this study indicate that providing 12 h of light during incubation can have a long-lasting effect on the diurnal rhythms of behavior, although the mechanism underlying this does not appear to be related to a persistent change in melatonin rhythm.

Diel changes in plasma cortisol and effects of size and stress duration on the cortisol response in European sea bass (Dicentrarchus labrax)

European sea bass (Dicentrarchus labrax), one of the most economically important fish in Mediterranean mariculture, shows high basal cortisol concentrations compared with other teleosts. The present study aims (a) to identify cortisol diel variation in fish held under a 12L:12D cycle and minimum handling stress, and (b) to establish the effect of fish size and stressor duration on the cortisol response. The results indicate high intrapopulation variability in plasma cortisol and a significant diel fluctuation with a peak value at dusk (18h). Stressors of different intensity and/or duration affected the cortisol stress response in a differential manner according to fish size (and/or age). Maximum cortisol values in small-size fish were found at 1 and 2h post-stress, depending on the duration of the stressor, while at 0.5h post-stress in large fish regardless stress duration.