Circadian time- and sleep-dependent modulation of cortical parvalbumin-positive inhibitory neurons.

Parvalbumin-positive neurons (PVs) are the main class of inhibitory neurons in the mammalian central nervous system. By examining diurnal changes in synaptic and neuronal activity of PVs in the supragranular layer of the mouse primary visual cortex (V1), we found that both PV input and output are modulated in a time- and sleep-dependent manner throughout the 24-h day. We first show that PV-evoked inhibition is stronger by the end of the light cycle (ZT12) relative to the end of the dark cycle (ZT0), which is in line with the lower inhibitory input of PV neurons at ZT12 than at ZT0. Interestingly, PV inhibitory and excitatory synaptic transmission slowly oscillate in opposite directions during the light/dark cycle. Although excitatory synapses are predominantly regulated by experience, inhibitory synapses are regulated by sleep, via acetylcholine activating M1 receptors. Consistent with synaptic regulation of PVs, we further show in vivo that spontaneous PV activity displays daily rhythm mainly determined by visual experience, which negatively correlates with the activity cycle of surrounding pyramidal neurons and the dorsal lateral geniculate nucleus-evoked responses in V1. These findings underscore the physiological significance of PV's daily modulation.

Circadian rhythm in turbot (Scophthalmus maximus): daily variation of blood metabolites in recirculating aquaculture systems.

INTRODUCTION: Animal welfare in aquaculture is becoming increasingly important, and detailed knowledge of the species concerned is essential for further optimization on farms. Every organism is controlled by an internal clock, the circadian rhythm, which is crucial for metabolic processes and is partially influenced by abiotic factors, making it important for aquaculture practices. OBJECTIVE: In order to determine the circadian rhythm of adult turbot (Scophthalmus maximus), blood samples were collected over a 24-h period and plasma metabolite profiles were analyzed by 1H-NMR spectroscopy. METHODS: The fish were habituated to feeding times at 9 am and 3 pm and with the NMR spectroscopy 46 metabolites could be identified, eight of which appeared to shift throughout the day. RESULTS: We noted exceptionally high values around 3 pm for the amino acids isoleucine, leucine, valine, phenylalanine, lysine, and the stress indicator lactate. These metabolic peaks were interpreted as either habituation to the usual feeding time or as natural peak levels in turbot in a 24-h circle because other indicators for stress (glucose, cortisol and lysozymes) showed a stable baseline, indicating that the animals had no or very little stress during the experimental period. CONCLUSION: This study provides initial insights into the diurnal variation of metabolites in adult turbot; however, further studies are needed to confirm present findings of possible fluctuations in amino acids and sugars. Implementing optimized feeding times (with high levels of sugars and low levels of stress metabolites) could lead to less stress, fewer disease outbreaks and overall improved fish welfare in aquaculture facilities.

Revisiting the Daily Timing of POS Phagocytosis.

Retinal pigment epithelium (RPE) cells daily ingest the tips of the photoreceptor outer segments (POSs), with phagosome number varying throughout a 24-h cycle. A major focus in the literature has been on a peak in phagosome concentration shortly after lights-on. Moreover, this peak has frequently been inferred to represent a peak in POS tip ingestion. Here, we have reviewed old and new literature on the daily cycle of phagosome number in the RPE and conclude that there is more variation in the timing of phagosome concentration peaks than is currently acknowledged. We also discuss that phagosome quantity is affected by the rate of phagosome degradation as well as the rate of ingestion; given that phagosome half-life may not be constant throughout the daily cycle, maximal POS ingestion may not necessarily coincide with a peak in phagosome concentration.

Effects of the timing of protein infusion on the daily rhythms of milk synthesis and plasma hormones and metabolites in dairy cows.

Milk synthesis exhibits a daily rhythm that is modified by the timing of feed intake. However, it is unknown how specific nutrients entrain this daily rhythm. Amino acids have an important role in milk synthesis, and may have a role in entrainment of mammary circadian rhythms. The objective of this study was to determine the effects of intestinally absorbed protein on daily rhythms of milk and milk component synthesis and key plasma hormones and metabolites. Nine lactating Holstein cows were assigned to 1 of 3 treatment sequences in a 3 × 3 Latin square. Treatments included abomasal infusions of 500 g/d of sodium caseinate either continuously throughout the day (CON), for 8 h/d from 0900 to 1700 h (DAY), or for 8 h/d from 2100 to 0500 h (NGT). Cows were milked every 6 h during the final 8 d of each period. A 24-h rhythm was fit to data using cosine analysis and the amplitude and acrophase were determined. Night infusion of protein decreased the daily milk yield and milk protein yield by 8.2% and 9.2%, respectively. Milk fat yield was increased 5.5% by DAY and milk fat concentration was increased 8.8% by NGT. Milk yield exhibited a daily rhythm in all treatments, with NGT increasing the amplitude of the daily rhythm 33% compared with CON. Milk fat concentration fit a daily rhythm in CON and NGT, but not DAY, whereas milk protein concentration fit a daily rhythm in CON and DAY, but not NGT. Moreover, DAY abolished the daily rhythm of plasma glucose concentration, but induced rhythms of plasma insulin and nonesterified fatty acid concentrations. Results suggest that feeding increased protein levels during the early part of the day may increase milk fat yield and modify energy metabolism through increased daily variation in insulin-stimulated lipid release, but additional research focused on feeding multiple diets across the day is required.

Critical symbiont signals drive both local and systemic changes in diel and developmental host gene expression.

The colonization of an animal's tissues by its microbial partners creates networks of communication across the host's body. We used the natural binary light-organ symbiosis between the squid Euprymna scolopes and its luminous bacterial partner, Vibrio fischeri, to define the impact of colonization on transcriptomic networks in the host. A night-active predator, E. scolopes coordinates the bioluminescence of its symbiont with visual cues from the environment to camouflage against moon and starlight. Like mammals, this symbiosis has a complex developmental program and a strong day/night rhythm. We determined how symbiont colonization impacted gene expression in the light organ itself, as well as in two anatomically remote organs: the eye and gill. While the overall transcriptional signature of light organ and gill were more alike, the impact of symbiosis was most pronounced and similar in light organ and eye, both in juvenile and adult animals. Furthermore, the presence of a symbiosis drove daily rhythms of transcription within all three organs. Finally, a single mutation in V. fischeri-specifically, deletion of the lux operon, which abrogates symbiont luminescence-reduced the symbiosis-dependent transcriptome of the light organ by two-thirds. In addition, while the gills responded similarly to light-organ colonization by either the wild-type or mutant, luminescence was required for all of the colonization-associated transcriptional responses in the juvenile eye. This study defines not only the impact of symbiont colonization on the coordination of animal transcriptomes, but also provides insight into how such changes might impact the behavior and ecology of the host.

Simultaneous recording of subcutaneous temperature and total locomotor activity in Bos taurus and Bos indicus raised in a subtropical region of Argentina.

The aim of this study was to evaluate some physiological parameters as total locomotor activity and subcutaneous temperature in two different bovine breeds under tropical environment. In this study, total locomotor activity and subcutaneous temperature were evaluated in 18 clinically healthy female cows. All cows were divided into 3 groups: group A1 was constituted by 6 Holstein Bos taurus, group A2 was constituted by 6 female Herford Bos taurus cows, and group B was constituted by 6 female Bradford Bos indicus cows. Data recording of total locomotor activity (TLA) and subcutaneous temperature (ST) was performed by means of a subcutaneous thermometer (Thermocron) and an actimeter ACTIWATCH® (Cambridge Neurotechnology Ltd.) based on accelerometer technologies equipped on all subjects to record TLA. At the same time, thermal and hygrometric recording were carried out, considering the subtropical climate of Santa Fe. The application of GLM for statistical analysis showed a significant effect (P < 0.05) on statistical model and time of the day on TLA and ST for all groups; no significative effects on animal parameters were found for ST and TLA except for group B. Circadian parameters have been evaluated according to the single cosinor procedure of ST that showed a diurnal daily rhythmicity for all investigated groups and TLA which is focused almost during the photophase for groups A1 and A2 and during scotophase for group B. Considering different species and breeds, and different environmental conditions, this study suggested that some subjects may be much more able to adapt themselves to environmental stress than others. During their evolution from Bos taurus, zebu cattle (Bos indicus) have acquired genes that confer thermotolerance. Subjects from Bos indicus breeds are better able to regulate body temperature in response to heat stress than Bos taurus subjects.

Impact of daytime light intensity on the central orexin (hypocretin) system of a diurnal rodent (Arvicanthis niloticus).

The neuropeptide orexin/hypocretin is implicated in sleep and arousal, energy expenditure, reward, affective state and cognition. Our previous work using diurnal Nile grass rats (Arvicanthis niloticus) found that orexin mediates the effects of environmental light, particularly daytime light intensity, on affective and cognitive behaviours. The present study further investigated how daytime light intensity affects the central orexin system in male and female grass rats. Subjects were housed for 4 weeks in 12:12 hr dim light:dark (50 lux, dimLD) or in 12:12 hr bright light:dark cycle (1000 lux, brightLD). Day/night fluctuations in some orexin measures were also assessed. Despite similar hypothalamic prepro-orexin mRNA expression across all conditions, there were significantly more orexin-immunoreactive neurons, larger somata, greater optical density or higher orexin A content at night (ZT14) than during the day (ZT2), and/or in animals housed in brightLD compared to dimLD. Grass rats in brightLD also had higher cisternal CSF levels of orexin A. Furthermore, orexin receptor OX1R and OX2R proteins in the medial prefrontal cortex were higher in brightLD than dimLD males, but lower in brightLD than dimLD females. In the CA1 and dorsal raphe nucleus, females had higher OX1R than males without any significant effects of light condition, and OX2R levels were unaffected by sex or light. These results reveal that daytime light intensity alters the central orexin system of both male and female diurnal grass rats, sometimes sex-specifically, and provides insight into the mechanisms underlying how daytime light intensity impacts orexin-regulated functions.

Reactive oxygen species homeostasis and circadian rhythms in plants.

Elucidation of the molecular mechanisms by which plants sense and respond to environmental stimuli that influence their growth and yield is a prerequisite for understanding the adaptation of plants to climate change. Plants are sessile organisms and one important factor for their successful acclimation is the temporal coordination of the 24 h daily cycles and the stress response. The crosstalk between second messengers, such as Ca2+, reactive oxygen species (ROS), and hormones is a fundamental aspect in plant adaptation and survival under environmental stresses. In this sense, the circadian clock, in conjunction with Ca2+- and hormone-signalling pathways, appears to act as an important mechanism controlling plant adaptation to stress. The relationship between the circadian clock and ROS-generating and ROS-scavenging mechanisms is still not fully understood, especially at the post-transcriptional level and in stress situations in which ROS levels increase and changes in cell redox state occur. In this review, we summarize the information regarding the relationship between the circadian clock and the ROS homeostasis network. We pay special attention not only to the transcriptional regulation of ROS-generating and ROS-scavenging enzymes, but also to the few studies that have been performed at the biochemical level and those conducted under stress conditions.

The daily gene transcription cycle in mouse retina.

Many physiological retinal processes, such as outer segment disk shedding and visual sensitivity, exhibit a daily rhythm. However, the detailed transcriptome dynamics and related biological processes of the retina are not fully understood. Retinal tissues were collected from C57BL/6J male mice housed in a 12h light/12h dark (LD) cycle for 4 weeks, at Zeitgeber time (ZT) 0, 4, 8, 12, 16, and 20. Total RNA was extracted from the tissues and used for unique identifier RNA sequencing experiments. The rhythmicity of gene expression was determined using the MetaCycle R package. We found that 1741 genes (10.26%) were rhythmically expressed in the retina. According to the expression patterns, the rhythmically expressed genes were assigned to four clusters, each with about 361-492 genes, using the Mfuzz R package. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were conducted to identify pathways and biological processes of the profiled genes. Genes in Clusters 1 and 4 were associated with glycolysis and energy production, showed higher activity at night (from ZT16 to ZT20), and were enriched in the Hif-1α signaling pathway and low-oxygen-related terms. Genes in Cluster 2 were predominantly involved in cilium assembly and organization and were relatively upregulated during the day. Genes in Cluster 3 were associated with ribosome biosynthesis and were highly expressed during the day-night transition period. Taken together, these results demonstrate that a large proportion of retinal genes are expressed rhythmically. Genes involved in energy production and glycolysis are highly expressed at night, leading to relative hypoxia and activation of the Hif-1α signaling pathway. Genes associated with the formation of photoreceptor cilia are expressed during the day.

The relationship between daily behavior, hormones, and a color dimorphism in a seabird under natural continuous light.

The predictable oscillation between the light of day and the dark of night across the diel cycle is a powerful selective force that has resulted in anticipatory mechanisms in nearly all taxa. At polar latitude, however, this oscillation becomes highly attenuated during the continuous light of polar day during summer. A general understanding of how animals keep time under these conditions is poorly understood. We tested the hypothesis that the common murre (a seabird, Uria aalge) can use melatonin and corticosterone, hormones associated with timekeeping, to track the diel cycle despite continuous light. We also tested the assumption that common murres breeding during polar summer schedule their colony attendance by time of day and sex, as they do at subpolar latitude. In the Atlantic population, common murres have a plumage color dimorphism associated with fitness-related traits, and we investigated the relationship of this dimorphism with colony attendance, melatonin, and corticosterone. The common murres did not schedule their attendance behavior by time of day or sex, yet they had higher concentrations of melatonin and, to a more limited extent, corticosterone during "night" than "day". Melatonin also linked to behavioral state. The two color morphs tended to have different colony-attendance behavior and melatonin concentrations, lending support for balancing selection maintaining the plumage dimorphism. In common murres, melatonin can signal time of day despite continuous light, and the limited diel variation of corticosterone contributes to the mounting evidence that polar-adapted birds and mammals require little or no diel variation in circulating glucocorticoids during polar day.

Daily and Estral Regulation of RFRP-3 Neurons in the Female Mice.

Female reproductive success relies on proper integration of circadian- and ovarian- signals to the hypothalamic-pituitary-gonadal axis in order to synchronize the preovulatory LH surge at the end of the ovarian follicular stage with the onset of the main active period. In this study, we used a combination of neuroanatomical and electrophysiological approaches to assess whether the hypothalamic neurons expressing Arg-Phe amide-related peptide (RFRP-3), a gonadotropin inhibitory peptide, exhibit daily and estrous stage dependent variations in female mice. Furthermore, we investigated whether arginine vasopressin (AVP), a circadian peptide produced by the suprachiamatic nucleus regulates RFRP-3 neurons. The number of c-Fos-positive RFRP-3 immunoreactive neurons is significantly reduced at the day-to-night transition with no difference between diestrus and proestrus. Contrastingly, RFRP neuron firing rate is higher in proestrus as compared to diestrus, independently of the time of the day. AVP immunoreactive fibers contact RFRP neurons with the highest density observed during the late afternoon of diestrus and proestrus. Application of AVP increases RFRP neurons firing in the afternoon (ZT6-10) of diestrus, but not at the same time point of proestrus, indicating that AVP signaling on RFRP neurons may depend on circulating ovarian steroids. Together, these studies show that RFRP neurons integrate both daily and estrogenic signals, which downstream may help to properly time the preovulatory LH surge.

Clock gene per 2 daily rhythm: Correlation with the serum level of uncoupling protein 1 (UCP1) in goat and horse.

Recent studies evidenced that the circadian rhythm of Per2 is involved in adaptive thermogenesis by the modulating transcription of uncoupling protein 1 (UCP1). For this purpose, we investigated the linkage between the daily rhythm of Per2 and UCP1 in ruminant and non-ruminant mammalian species. Five clinically healthy, not pregnant, and not lactating Maltese female goats and five clinically healthy, not pregnant, and not lactating Italian Saddle horses were enrolled in the study. All animals were housed under natural photoperiod (sunrise 05:05, sunset 20:55) and environmental temperature and humidity. Goats were kept individually in 3.0 × 2.0 m box, horses were housed individually in 3.5 × 3.5 m box; all boxes were equipped with an opening window. On each subject, blood samples were collected every 4 h for a 48-h period. The Per2 gene expression was determined on blood samples collected in PAX gene Blood RNA Tube, whereas UCP1 concentration was assessed on serum. Per2 and UCP1 levels were statistically influenced by the species (p < 0.0001) and the time of data collection (p < 0.0001), but not by the day of monitoring. Per2 showed daily rhythmicity, statistically different in mesor and amplitude between the two species, diurnal in goats, nocturnal in horses; with the same robustness. UCP1 did not show daily rhythmicity. During the experimental period the two parameters showed a negative correlation in horses. According to the findings herein obtained, we can claim that the role of Per2 in the thermogenesis induced by the beige adipocytes throughout UCP1 activation did not reflect what found in other mammal species, but further studies are required to establish their correlation in equids.

A Kiss to drive rhythms in reproduction.

Reproduction, like many other biological functions, exhibits marked daily and seasonal rhythms in order to anticipate and adapt breeding activity to environmental challenges. In recent years, studies investigating the neuroendocrine mechanisms driving rhythms in reproduction have unveiled the pivotal role of hypothalamic neurons expressing kisspeptin in integrating and forwarding daily and seasonal cues to the reproductive system. The objective of this review is to summarize the knowledge on the effect and role of this neuropeptide on the mammalian hypothalamo-pituitary-gonadal axis and describe how it is involved in the daily control of ovulation in females and long-term adaptation of reproduction in seasonal breeders.

Hypothalamic leptin sensitivity and health benefits of time-restricted feeding are dependent on the time of day in male mice.

Synchronization between biologic clocks and metabolism is crucial for most species. Here, we examined the ability of leptin, important in the control of energy metabolism, to induce leptin signaling at the molecular as well as the behavioral level throughout the 24-h day in mice fed either a control or a high-fat diet (HFD). Furthermore, we investigated the effects of time-restricted feeding (TRF; a limitation of HFD access to 6 h each day) on energy metabolism during different periods throughout the 24-h day. In control mice, molecular leptin sensitivity was highest at zeitgeber time (ZT)0 (lights on), declining during the light phase, and increasing during the dark phase. Surprisingly, leptin resistance in HFD-fed mice was only present from the middle of the dark to the middle of the light period. Specifically, when TRF occurred from ZT21 to ZT3 (when leptin resistance in HFD-fed mice was most profound), it resulted in a disruption of the daily rhythms of locomotor activity and energy expenditure and in increased plasma insulin levels compared with other TRF periods. These data provide evidence that leptin sensitivity is controlled by the circadian rhythm and that TRF periods may be most efficient when aligned with the leptin-sensitive period.-Boucsein, A., Rizwan, M. Z., Tups, A. Hypothalamic leptin sensitivity and health benefits of time-restricted feeding are dependent on the time of day in male mice.

Lower activity of salivary alpha-amylase in youths with depression.

Measurement of basal and stress-induced salivary alpha-amylase activity may help to understand autonomic nervous system disturbance in mental disorders. The potential sympathetic nervous system dysregulation in children and adolescent psychopathologies is mostly unknown. The present study was aimed to test the hypothesis that salivary alpha-amylase activity is higher in youths diagnosed with depression than in healthy subjects considering a part of the daily rhythm of enzyme activity and its morning to midday slope. A total of 30 children aged 15 ± 0.46 years (15 patients with depression, 4 boys, 11 girls, and 15 sex- and age-matched healthy controls) participated in the study. Two saliva samples were collected from each subject to measure activity of alpha-amylase in the morning and midday. The results of the present study revealed that the midday but not morning alpha-amylase activity was lower in patients with depression than in healthy controls. The diurnal increase in enzyme activity present in healthy subjects was absent in patients. The children and adolescents with depression exhibited flatter morning to midday slopes of alpha-amylase activity. In conclusion, the present results indicate a disturbance of alpha-amylase daily rhythm in youths with depression and motivate further studies on the relationship between sympathetic activation and mood disorders.

Night-restricted feeding of dairy cows modifies daily rhythms of feed intake, milk synthesis and plasma metabolites compared with day-restricted feeding.

The timing of feed intake can alter circadian rhythms of peripheral tissues. Milk synthesis displays a daily rhythm across several species, but the effect of feeding time on these rhythms is poorly characterised. The objective of this experiment was to determine if the time of feed intake modifies the daily patterns of milk synthesis, plasma metabolites and body temperature in dairy cows. Sixteen lactating Holstein dairy cows were randomly assigned to one of the two treatment sequences in a cross-over design with 17 d periods. Treatments included day-restricted feeding (DRF; feed available from 07.00 to 23.00 hours) and night-restricted feeding (NRF; feed available from 19.00 to 11.00 hours). Cows were milked every 6 h on the last 7 d of each period, and blood samples were collected to represent every 4 h over the day. Peak milk yield was shifted from morning in DRF to evening in NRF, while milk fat, protein and lactose concentration peaked in the evening in DRF and the morning in NRF. Plasma glucose, insulin, NEFA and urea nitrogen concentration fit daily rhythms in all treatments. Night feeding increased the amplitude of glucose, insulin and NEFA rhythms and shifted the daily rhythms by 8 to 12 h (P < 0·05). Night feeding also phase-delayed the rhythm of core body temperature and DRF increased its amplitude. Altering the time of feed availability shifts the daily rhythms of milk synthesis and plasma hormone and metabolite concentrations and body temperature, suggesting that these rhythms may be entrained by food intake.

Daily variation of D2 dopamine receptor transcription in the brain of the Japanese eel Anguilla japonica and its regulation with dopamine and melatonin.

Dopamine plays a crucial role in controlling reproduction in eels, and its action is mediated through D2-type dopamine receptors. D2A and D2B receptors in the Japanese eel Anguilla japonica were cloned and characterized in the present study. Attention (daily expression patterns in the brain and endogenous regulation) was paid to D2B receptor because it is considered to play a crucial role in eel reproduction. The cDNAs of D2A and D2B receptors had open reading frames comprising 456 and 454 amino acid residues, respectively, which were phylogenetically clustered with those of other teleost species. Both receptors were highly expressed in the brain. D2B receptor transcript levels exhibited high day/low night variation in the midbrain and pituitary, suggesting that its transcription in these tissues is regulated in a daily manner, possibly under influence of melatonin. Intraperitoneal injection of dopamine downregulated D2B receptor transcription significantly in the midbrain and moderately in the pituitary within 1 h, but upregulated its transcription in the forebrain. Co-injection of dopamine with its antagonist (domperidone) reversed the effect of dopamine in the pituitary and forebrain, but not in the midbrain, suggesting that the effect of dopamine on D2B receptor transcription differs among brain regions. The same treatment with melatonin resulted in decreased D2B receptor transcription in the midbrain. These findings indicate that dopamine and melatonin have key roles in the daily variation in D2B receptor transcription in the brain of Japanese eel, and that they are related to a daily base secretion of hormones in the hypothalamic-pituitary-gonadal axis in this species.

Sampling strategy and measurement device affect vaginal temperature outcomes in lactating dairy cattle.

Body temperature (BT) is widely used to evaluate health and heat load status in cattle. Despite its importance, studies vary in how BT is measured and in the biological interpretation of the results. Costs, practicality, labor, and welfare concerns can affect how BT is measured, including frequency of measurement and the type of device used. Inaccurate BT outcomes may have implications for cattle welfare; for example, animals may only receive treatment when fever is identified. Our objectives were (1) to compare measurement of vaginal temperature (VT) using relatively small, inexpensive, and low-accuracy loggers (±0.5 to ±1°C, iButton range; Embedded Data Systems, Lawrenceburg, KY) to a high-accuracy logger (±0.1°C; StarOddi, Gardabaer, Iceland), and (2) to evaluate how different BT sampling strategies correspond to 24-h VT in lactating dairy cows. To address the first objective, VT data from 54 cows were recorded every 45 min for 12 d/cow, on average, using 2 different types of temperature loggers (StarOddi DST centi-T and iButton DS1921H or DS1922L) attached to a shortened, hormone-free controlled internal drug release insert. Average VT obtained from both loggers were compared using mixed models and regression analyses. In addition, we tested the consistency of the low-accuracy loggers in detecting cows with elevated BT using the kappa coefficient of concordance. To address the second objective, VT data from 20 cows were recorded every min for 9 to 11 d/cow using StarOddi loggers. Using these data, we estimated average VT using 11 sampling strategies (every 5, 10, 15, 30, 45, 60, and 120 min, 1×/d recorded in the morning or afternoon, 2×/d, or 3×/d). Estimates and observed means were compared using linear regression. Compared with StarOddi loggers, the iButtons either underestimated (H model: 38.7 vs. 38.0 ± 0.06°C) or overestimated VT (L model: 38.7 vs. 39.2 ± 0.04°C). When considering elevated or fever VT thresholds, iButtons did not correctly classify animals; kappa coefficients of concordance were ≤0.35. Measuring VT as often as every 120 min resulted in more accurate estimates compared with strategies that recorded it once to thrice per day. These results indicate that the type of device (i.e., data logger) and sampling strategies affect BT outcomes and that these decisions affect the interpretation of BT data.

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

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

Daily expression of a clock gene in the brain and pituitary of the Malabar grouper (Epinephelus malabaricus).

Recent studies have revealed that, in addition to regulating the circadian system, clock genes such as cryptochrome (Cry) genes are involved in seasonal and lunar rhythmicity in fish. This study clarified the transcriptional characteristics of a Cry subtype (mgCry2) in the brain of the Malabar grouper, Epinephelus malabaricus, which is an important aquaculture species that spawns around the new moon. The cDNA sequence of mgCry2 showed high identity (97-99%) with fish Cry2 and had an open reading frame encoding a protein with 170 amino acids. Phylogenetic analyses revealed that mgCRY2 had high identity with CRY in other fish species. Real-time quantitative polymerase chain reaction (qPCR) showed the widespread distribution of mgCry2 in neural (brain, pituitary, and retina) and peripheral (heart, liver, kidney, spleen, gill, intestine, and ovary) tissues. When immature Malabar groupers were reared under a light-dark cycle (LD = 12:12) and the amounts of mgCry2 mRNA in the telencephalon and diencephalon were measured at 4-h intervals, the levels increased during photophase and decreased during scotophase. Day-night variation in mgCry2 mRNA abundance was also observed in the pituitary. These daily profiles suggest that mgCry2 is a light-responsive gene in neural tissues. In situ hybridization analyses showed that mgCry2 was strongly transcribed in the nucleus lateralis tuberis of the ventral hypothalamus, peripheral area of the proximal pars distalis, and the pars intermedia of the pituitary. We conclude that clock genes expressed in the pituitary and diencephalon play a role in entraining the endocrine network of the Malabar grouper to periodic changes in external cues.