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It is known that the level of dietary protein modulates the enzymatic activity of the digestive tract of fish; however, its effect at the molecular level on these enzymes and the hormones regulating appetite has not been well characterised. The objective of this study was to evaluate the effect of CP on the activity of proteases and the expression of genes related to the ingestion and protein digestion of juveniles of red tilapia (Oreochromis sp.), as well as the effects on performance, protein retention and body composition of tilapia. A total of 240 juveniles (29.32 ± 5.19 g) were used, distributed across 20 tanks of 100 l in a closed recirculation system. The fish were fed to apparent satiety for 42 days using four isoenergetic diets with different CP levels (24%, 30%, 36% and 42%). The results indicate that fish fed the 30% CP diet exhibited a higher growth performance compared to those on the 42% CP diet (P < 0.05). Feed intake in fish fed 24% and 30% CP diets was significantly higher than that in fish fed 36% and 42% CP diets (P < 0.05). A significant elevation of protein retention was observed in fish fed with 24% and 30% CP diets. Fish fed with 24% CP exhibited a significant increase in lipid deposition in the whole body. The diet with 42% CP was associated with the highest expression of pepsinogen and the lowest activity of acid protease (P < 0.05). The expression of hepatopancreatic trypsinogen increased as CP levels in the diet increased (P < 0.05) up to 36%, whereas trypsin activity showed a significant reduction with 42% CP (P < 0.05). The diet with 42% CP was associated with the lowest intestinal chymotrypsinogen expression and the lowest chymotrypsin activity (P < 0.05). α-amylase expression decreased with increasing (P < 0.05) CP levels up to 36%. No significant differences were observed in the expression of procarboxypeptidase, lipase or leptin among all the groups (P > 0.05). In addition, the diet with 42% CP resulted in a decrease (P < 0.05) in the expression of ghrelin and insulin and an increase (P < 0.05) in the expression of cholecystokinin and peptide yy. It is concluded that variation in dietary protein promoted changes in the metabolism of the red tilapia, which was reflected in proteolytic activity and expression of digestion and appetite-regulating genes.

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Protein is the most costly nutrient in fish feed, and while diets offered in the early stages of development typically have high levels of CP, they do not always correspond to the real requirements of the animals. Thus, research that seeks to learn the true nutritional requirements of fish is fundamental to improving commercial fish culture. The present study evaluated the protein requirements of Nile tilapia (Oreochromis niloticus) under larviculture. Fish performance, gene expression for digestive enzymes and their enzymatic activity and stress response to air exposure were analyzed. Four experimental diets differing in CP level were formulated: 30%, 36%, 42% and 48%. Fish larvae were fed the experimental diets during development and sampled 10, 20 and 30 days after the beginning of the experiment for performance, gene expression and enzymatic activity. At sampling time 30, stress resistance was also evaluated by means of an air exposure test. At sampling time 10, CP levels between 36% and 48% could be used for a better performance. During this period, pepsinogen expression was greater for 30% CP, intermediate for 42% and lower for 36% and 48%. After this initial period, diets of between 30% and 42% CP are recommended for better performance. At sampling time 20, gene expression for digestive enzymes and their enzymatic activity were similar for all diets tested. At sampling time 30, the diet of 42% CP induced both greater pepsinogen expression and pepsin activity. Survival after the air exposure test after 30 days of feeding was influenced by CP level in the diet, with the highest survival being for fish fed with 36% CP. Taken together, the present results demonstrate that dietary CP influences digestive enzyme gene expression and activity, and suggest that the best CP levels for Nile tilapia larviculture vary depending on larval stage.

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Growth factors in vertebrates display daily rhythms, which, while widely described in mammals, are still poorly understood in teleost fish. Here, we investigated the existence of daily rhythms in the somatotropic axis of the flatfish Solea senegalensis. In a first experiment, daily rhythms of the expression of pituitary adenylate cyclase-activating polypeptide (pacap), growth hormone (gh), insulin-like growth factor 1 (igf1) and its receptor (igf1r) were analyzed under a 12:12 h light:dark cycle. All genes displayed daily rhythms with the acrophases of pacap, gh and igf1 located in the second half of the dark phase (ZT 20:28-0:04 h), whereas the acrophase of igf1r was located around mid-light (ZT 5:33 h). In a second experiment, the influence of the time of day (mid-light, ML, versus mid-darkness, MD) of GH administration on the expression of these factors and on plasma glucose levels was tested. The response observed depended on the time of injection: the strongest effects were observed at MD, when GH administration significantly reduced pituitary gh and enhanced liver igf1 expression. These results provide the first evidence of daily rhythms and differential day/night effects in growth factors in S. senegalensis, suggesting new insights for investigating the physiology of growth and possible applications to improve fish aquaculture.

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Despite numerous studies about fish nutrition and lipid metabolism, very little is known about the daily rhythm expression of lipogenesis and lipolysis genes. This research aimed to investigate the existence of daily rhythm expressions of the genes involved in lipid metabolism and their synchronization to different light/dark (LD) and feeding cycles in zebra fish liver. For this purpose, three groups of zebra fish were submitted to a 12:12 h LD cycle. A single daily meal was provided to each group at various times: in the middle of the light phase (ML); in the middle of the dark phase (MD); at random times. After 20 days of acclimation to these experimental conditions, liver samples were collected every 4 h in one 24-h cycle. The results revealed that most genes displayed a significant daily rhythm with an acrophase of expression in the dark phase. The acrophase of lipolytic genes (lipoprotein lipase - lpl, peroxisome proliferator-activated receptor - pparα and hydroxyacil CoA dehydrogenase - hadh) was displayed between ZT 02:17 h and ZT 18:31 h. That of lipogenic genes (leptin-a - lepa, peroxisome proliferator-activated receptor - pparγ, liver X receptor - lxr, insulin-like growth factor - igf1, sterol regulatory element-binding protein - srebp and fatty acid synthase - fas) was displayed between ZT 15:25 h and 20:06 h (dark phase). Feeding time barely influenced daily expression rhythms, except for lxr in the MD group, whose acrophase shifted by about 14 h compared with the ML group (ZT 04:31 h versus ZT 18:29 h, respectively). These results evidence a strong synchronization to the LD cycle, but not to feeding time, and most genes showed a nocturnal acrophase. These findings highlight the importance of considering light and feeding time to optimize lipid metabolism and feeding protocols in fish farming.

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The endocrine axis controlling the stress response displays daily rhythms in many factors such as adrenal sensitivity and cortisol secretion. These rhythms have mostly been described in mammals, whereas they are poorly understood in teleost fish, so that their impact on fish welfare in aquaculture remains unexplored. In the present research, the authors investigated the daily rhythms in the hypothalamus-pituitary-interrenal (HPI) axis in the flatfish Solea senegalensis, which has both scientific and commercial interest. In a first experiment, hypothalamic expression of corticotropin-releasing hormone (crh) and its binding protein (crhbp), both pituitary proopiomelanocortin A and B (pomca and pomcb) expression, as well as plasma cortisol, glucose, and lactate levels were analyzed throughout a 24-h cycle. All variables displayed daily rhythms (cosinor, p < .05), with acrophases varying depending on the factor analyzed: crh and cortisol peaked at the beginning of the dark phase (zeitgeber time [ZT] = 14.5 and 14.4 h, respectively), pomca and pomcb as well as glucose at the beginning of the light phase (ZT = 1.2, 2.4, and 3.4 h, respectively), and crhbp and lactate at the end of the dark phase (ZT = 22.3 and 23.0 h, respectively). In a second experiment, the influence of an acute stressor (30 s of air exposure), applied at two different time points (ZT 1 and ZT 13), was tested. The stress response differed depending on the time of day, showing higher cortisol values (96.2 ± 10.7 ng/mL) when the stressor was applied at ZT 1 than at ZT 13 (52.6 ± 11.1 ng/mL). This research describes for the first time the daily rhythms in endocrine factors of the HPI axis of the flatfish S. senegalensis, and the influence of daytime on the stress responses. A better knowledge of the chronobiology of fish provides a helpful tool for understanding the circadian physiology of the stress response, and for designing timely sound protocols to improve fish welfare in aquaculture.

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The effect of intracoelomic tagging of an acoustic telemetry transmitter (1.65% ratio of tag mass in the air to fish mass in the air) on behavioral (food intake and locomotor activity) and physiological (blood glucose and plasma cortisol) parameters of gilthead seabream was investigated. To this end, fish (289 ± 53 g, mean ± SD) were divided into 3 experimental groups: control (C), transmitter (T, inserted surgically) and sham group (S, subjected to surgery but without transmitter insertion). Blood was extracted during surgery and 9 days later. Throughout the trial, fish were fed by means of self-feeders and locomotor activity was measured by means of an infrared photocell. Two days after the first manipulation, a significant decrease in food intake could be observed in all the experimental groups. The fact that food intake was not affected after the second manipulation seems to indicate that a learning process took place for handling. The rhythmicity of feeding and locomotor activity was not affected by handling in any experimental group. However, group T showed increased plasma cortisol levels 9 days after surgery. In conclusion, while most behavioral parameters were not affected by handling, the plasma cortisol levels of seabream 9 days after insertion of the transmitter indicated a physiological impact that should be taken into account in long-term radiotracking studies, since such an operation could have negative effects on wild individuals after the end of the tracking experiments.

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Increased aquaculture production has raised concerns about managing protocols to safeguard the welfare of farmed fish, as consumers demand responsible aquaculture practices to provide 'welfare friendly' products. Feeding is one of the largest production cost in a fish farm and can be one of the biggest stressors for fish. Under farming conditions, fish are challenged with artificial diets and feeding regimes, and inadequate feeding conditions cause stress, alteration of normal behavioural patterns, poor performance and eventually diseases and death, which are by no means acceptable neither economically nor ethically. This review aims to highlight the impact of feeding rhythms and feeding time upon physiological and behavioural welfare indicators, which show circadian rhythms as well. Therefore, all these variables should be considered when designing feeding strategies in farming conditions and assessing the welfare state of cultured fish.

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Food availability is far from constant but tends to be cyclic, and fish therefore show a variety of circadian rhythms which can be entrained to feeding time. The aim of this study was to investigate the synchronization to mealtimes of behavioral (locomotor activity), metabolic (glucose) and endocrine (cortisol and thyroid hormones) daily rhythms in gilthead seabream. To this end, fish were reared under a 12:12 LD cycle and fed 1% of their body weight once a day either at mid-light (ML) or at mid-dark (MD) of the LD cycle. Fish synchronized their locomotor activity to the phase in which food was delivered, ML and MD fish displaying 86+/-3% and 81+/-1% of their total daily activity during daytime and nighttime, respectively. Daily variations of blood glucose were strongly synchronized to feeding time in both experimental groups, peaking 8h after the meal. A postprandial cortisol peak was observed in both groups. In fish fed at MD the cortisol values were high during the 8h following feeding, whereas in fish fed at ML cortisol levels returned to basal values within 4h. In addition, MD fish showed a higher average daily cortisol value (24.75+/-2.65 ng/ml) than ML fish (10.30+/-2.18 ng/ml). Feeding time affected the magnitude of daily variations in the thyroid hormones. When the time of feeding was delayed by 12h, a shift of the same magnitude could be observed in the glucose daily rhythm and a dramatic change in the cortisol levels of the ML-fed group compared with results mentioned above. In summary, gilthead seabream synchronized their locomotor activity to the phase when food was provided and showed different degrees of synchronization of their behavioral, metabolic and endocrine rhythms to feeding time. Since these parameters are used to evaluate stress responses and welfare in seabream, their daily rhythm and synchronization to light and feeding time should be taken into account.

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Feeding cycles entrain biological rhythms, which enable animals to anticipate feeding times and so maximize food utilization and welfare. In this article, the effect of mealtime was investigated in two groups of sea bream (Sparus aurata): one group received a single daily meal at random times during the light period (random daytime feeding, RDF), whereas the other group received the meal during the light period but at the same time (scheduled daytime feeding, SDF). All the fish showed diurnal behavior, although the SDF group showed a lower percentage of diurnalism (84.4% vs. 79.5% in RDF and SDF respectively) and developed food anticipatory activity some hours before the mealtime. In addition, the mean daily locomotor activity of the RDF group was significantly higher than that of the SDF group (3132 vs. 2654 counts/day, respectively). Although the mean weight differed between both groups on day 30 (115.7 g and 125.6 g in RDF and SDF respectively), these differences had disappeared by day 60. Plasma cortisol and glucose significantly differed in both groups (cortisol: 71.8 vs. 8.7 ng/ml, glucose: 53.7 vs. 43.8 mg/dl in RDF and SDF, respectively), whereas lactate did not differ significantly. The results obtained suggest that altering the feeding time (scheduled vs. random) affects the behavior and physiology of sea bream, indicating that a single daily feeding cycle (compared to random) is beneficial for fish welfare because they can prepare themselves for the forthcoming feed.

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Daily rhythms in glucose tolerance have been reported for several species of mammals, which seem to be linked to factors such as daily rhythms of insulin resistance and feeding habits. In this paper, we studied entrainment to a daily scheduled meal of blood glucose after carbohydrate intake (dextrin or glucose). After a meal containing dextrin, blood glucose showed different maximum concentrations (Cmax) and time to peak (Tmax) depending on mealtime, the greatest differences in Cmax being found 2 h after feeding. The highest Cmax (8.22 mmol/l) was obtained when mealtime was set in the middle of the light phase (ML), and the lowest Cmax (3.46 mmol/l) when goldfish were fed in the middle of the dark phase (MD). Cosinor analysis revealed a significant daily rhythm in dextrin tolerance with its acrophase around ML, amplitude of 1.99 mmol/l, and a mesor of 5.49 mmol/l. However, when the carbohydrate source in the meal was glucose, an inverse pattern was observed: higher blood glucose in goldfish fed at MD compared with ML (6.98 vs 4.32 mmol/l, respectively). Similar results were obtained when glucose was administered intraperitoneally, with higher values at MD than at ML (13.82 vs 9.54 mmol/l, respectively). Finally, no differences in amylase activity were observed in the gut after dextrin load at ML and MD (522 vs 446 U/mg protein), discarding the effect of digestive factors in the daily rhythm of tolerance. In conclusion, our results described for the first time a daily rhythm in tolerance to carbohydrate in a teleost fish, highlighting the impact of the time of day in glucose metabolism.

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Sea bass is an euryhaline fish that lives in a wide range of salinities and migrates seasonally from lagoons to the open sea. However, to date, the influence of water salinity on sea bass melatonin levels has not been reported. Here, we evaluated the differences in plasma and tissue melatonin contents and melatonin binding sites in sea bass under four different salinity levels: seawater (36 per thousand), isotonic water (15 per thousand), brackish water (4 per thousand) and freshwater (0 per thousand). The melatonin content was evaluated in plasma, whole brain, gills, intestine and kidney, while melatonin binding sites were analyzed in different brain regions and in the neural retina. Plasma melatonin levels at mid-dark varied, the lowest value occurring in seawater (102 pg/mL), and the highest in freshwater (151 pg/mL). In gills and intestine, however, the highest melatonin values were found in the seawater group (209 and 627 pg/g tissue, respectively). Melatonin binding sites in the brain also varied with salinity, with the highest density observed at the lower salinities in the optic tectum, cerebellum and hypothalamus (30.3, 13.0, and 8.0 fmol/mg protein, respectively). Melatonin binding sites in the retina showed a similar pattern, with the highest values being observed in freshwater. Taken together, these results reveal that salinity influences melatonin production and modifies the density of binding sites, which suggests that this hormone could play a role in timing seasonal events in sea bass, including those linked to fish migration between waters of different salinities for reproduction and spawning.

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The melatonin daily rhythm provides the organism with photoperiod-related information and represents a mechanism to transduce information concerning time of day. In addition, the duration and amplitude of the nocturnal elevation gives information about duration and thus the time of year. In this study, we investigate the existence of an annual rhythm of plasma melatonin in the Senegal sole. Differences in plasma melatonin levels between fish kept at a controlled temperature (17-20 degrees C) and those exposed to the environmental temperature cycle (11.5-25 degrees C) were also examined throughout the year. Spawning was registered in both groups to determine the time of year in which reproductive rhythms occurred. Our results pointed to the existence of an annual rhythm of plasma melatonin at mid-darkness (MD), with the highest levels (203 +/- 44 pg/mL) observed when water temperature reached 25 degrees C. Water temperature influenced nocturnal, but not diurnal melatonin. Daily melatonin rhythms showed seasonal differences, with higher mean nocturnal levels during the summer solstice (138 +/- 19 pg/mL) and autumn equinox (149 +/- 49 pg/mL). When animals were kept at a constant temperature throughout the year, plasma melatonin levels differed from those observed in fish exposed to the environmental temperature cycle. Regarding the reproductive rhythms, spawning was observed at the end of spring in sole kept under natural temperature conditions, whereas no spawning at all was registered in sole reared at a constant temperature. In short, both photoperiod and temperature affected melatonin production in the Senegal sole, transducing seasonal information and controlling annual reproductive rhythms.

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Melatonin has a number of physiological functions in addition to light-dark transduction. In recent years, many in vivo and in vitro studies in rodents have revealed an important antioxidant activity of melatonin, both directly and indirectly. Nevertheless, the potential effects of melatonin as an antioxidant in fish remain unknown. The aim of this research was to evaluate the capacity of melatonin injections (3 mg/kg) to attenuate oxidative damage after submitting goldfish to oxidative stress caused directly by hydrogen peroxide (H2O2) baths and indirectly by hypoxia and subsequent reoxygenation, as well as the locomotor activity. The results revealed that melatonin decreased lipid damage in muscle after hypoxia/reoxygenation (1.22 vs. 2.27 nmoles lipid peroxides/g tissue), but not in liver. Mortality caused by oxidative stress was not attenuated by melatonin. Surprisingly, melatonin caused an increase of mortality (50 vs. 95%) when administered before hypoxia. Locomotor activity was also affected by melatonin but not by the administration of the vehicle, suggesting a sedative effect of melatonin in goldfish. In conclusion, melatonin administration provoked slight effects on lipid peroxidation and mortality resulting from oxidative stress, with reduction of locomotor activity in relation to the vehicle.

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Melatonin has several known physiological functions, the main one being synchronization of daily and seasonal rhythms. In addition, melatonin has been reported to influence food intake and behavioral rhythms with varying results depending on the species. The aim of this research was to evaluate the effects of intraperitoneal melatonin injection on food intake and locomotor activity in two different fish species: goldfish (diurnal) and tench (nocturnal), under different light regimes: constant light (LL) conditions or LD 12:12, with melatonin administration at mid-light (ML), mid-dark (MD), and after a 1-h light pulse at MD. In addition to these acute tests, in the case of goldfish we also investigated the effects of daily melatonin administration for 1 week. Our results indicated that acute melatonin administration significantly decreased goldfish food intake (16-52% inhibition, depending on the light regime) and locomotor activity (55-100%), with the chronic treatment inducing a similar total food intake inhibition that persisted for 7 days. In tench, a nocturnal fish species, acute melatonin administration at MD and ML reduced food intake (37% and 29%, respectively), while locomotor activity was not affected at MD and slightly increased at ML. Taken together, these results indicated that melatonin reduced food intake in both species, while its effects on locomotor activity depended on the time of administration (light or dark phase) and the activity patterns of the species.

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Melatonin has a number of physiological functions in addition to light-dark transduction.In recent years, many in vivo and in vitro studies in rodents have revealedan important antioxidant activity of melatonin, both directly and indirectly. Nevertheless,the potential effects of melatonin as an antioxidant in fish remain unknown.The aim of this research was to evaluate the capacity of melatonin injections (3mg/kg) to attenuate oxidative damage after submitting goldfish to oxidative stresscaused directly by hydrogen peroxide (H2O2) baths and indirectly by hypoxia andsubsequent reoxygenation, as well as the locomotor activity. The results revealed thatmelatonin decreased lipid damage in muscle after hypoxia/reoxygenation (1.22 vs2.27 nmoles lipid peroxides/g tissue), but not in liver. Mortality caused by oxidativestress was not attenuated by melatonin. Surprisingly, melatonin caused an increase ofmortality (50 vs 95%) when administered before hypoxia. Locomotor activity wasalso affected by melatonin but not by the administration of the vehicle, suggesting asedative effect of melatonin in goldfish. In conclusion, melatonin administration provokedslight effects on lipid peroxidation and mortality resulting from oxidativestress, with reduction of locomotor activity in relation to the vehicle

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Melatonin production by the pineal organ is influenced by light intensity, as has been described in most vertebrate species, in which melatonin is considered a synchronizer of circadian rhythms. In tench, strict nocturnal activity rhythms have been described, although the role of melatonin has not been clarified. In this study we investigated daily activity and melatonin rhythms under 12:12 light-dark (LD) conditions with two different light intensities (58.6 and 1091 microW/cm2), and the effect of I h broad spectrum white light pulses of different intensities (3.3, 5.3, 10.5, 1091.4 microW/cm2) applied at middarkness (MD) on nocturnal circulating melatonin. The results showed that plasma melatonin in tench under LD 12:12 and high light conditions displayed rhythmic variation, where values at MD (255.8 +/- 65.9 pg/ml) were higher than at midlight (ML) (70.7 +/- 31.9 pg/ml). Such a difference between MD and ML values was reduced in animals exposed to LD 12: 12 and low light intensity. The application of 1 h light pulses at MD lowered plasma melatonin to 111.6 +/- 3.2 pg/ml (in the 3.3-10.5 microW/cm2 range) and to 61.8 +/- 18.3 pg/ml (with the 1091.4 microW/cm2 light pulse) and totally suppressed nocturnal locomotor activity. These results show that melatonin rhythms persisted in tench exposed to low light intensity although the amplitude of the rhythm is affected. In addition, it was observed that light pulses applied at MD affected plasma melatonin content and locomotor activity. Such a low threshold suggests that the melatonin system is capable of transducing light even under dim conditions, which may be used by this nocturnal fish to synchronize to weak night light signals (e.g., moonlight cycles).

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The daily locomotor and melatonin rhythms of the Senegal sole, a benthonic species of increasing interest in aquaculture, are still unknown, despite the fact that such knowledge is of prime importance for optimising its production. The aim of the present research was therefore to investigate the daily rhythms of locomotor activity and melatonin in the Senegal sole. For this purpose, the individual locomotor activity rhythms of fish were registered using a photocell. Plasma and ocular melatonin rhythms were studied in animals reared in circular tanks placed in earth under an LD 12:12 light regime and 16-18 degrees C temperature range (spring equinox). Blood and eye samples were taken every 3 h during a complete 24-h cycle. The impact of a light pulse in the middle of the dark period (MD) on plasma melatonin was also studied. Locomotor activity was mainly nocturnal, with 84.3% of the total activity occurring during darkness. The levels of plasma melatonin were higher at night (55 pg/ml) than during the day (2 pg/ml), while ocular melatonin levels appeared to be arrhythmic. Both weight and melatonin content were found to be significantly higher in the left eye in relation to the right eye. A light pulse in MD provoked a significant decrease in plasma melatonin levels. In summary, photoperiod is a key factor in synchronizing locomotor activity and melatonin rhythms in the Senegal sole, whose nocturnal habits should be taken into account for their rearing by aquaculture.

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