Daily rhythms of swimming activity,synchronization to different feeding times and effects on anesthesia practice in an Amazon fish species (Colossoma macropomum)

ABSTRACT

This study aimed to investigate the existence of day-night differences in the time for anesthesia and recovery in tambaqui exposed to the anesthetic eugenol and the influence of feeding time. Thus, we evaluated: (1) swimming activity; (2) food anticipatory activity (FAA) as a synchronizer of swimming activity and change to susceptibility to anesthetic; and (3) the effects of diurnal/nocturnal anesthesia exposure of fish feeding in the mid-light phase: 12:00 h (ML) and fish feeding in the mid-dark phase: 00:00 h (MD). Our findings revealed strictly nocturnal activity for tambaqui (94.2%), known as diurnal fish to date. Moreover, FAA was observed in tambaqui fed at MD, which showed a sustained increase in activity that began 2 h before feeding time and lasted until feeding. In contrast, no FAA was observed in fish fed at ML. Regarding anesthesia by day or night, the tambaqui treated with eugenol exhibited no difference in induction time. However, differences were observed in recovery times, with fish anesthetized at day recovering in 1–2 min and fish anesthetized at night recovering in 5–7 min. In short, our findings revealed for the first time the nocturnal behavior of tambaqui. These results indicated that recovery by day/night by eugenol in tambaqui has a strong dependence of behavioral patterns and the time of day.     

Influence of light on feeding anticipatory activity in goldfish

Most animals when presented with food on a periodic basis develop feeding anticipatory activity (FAA). The objective of the present study was to investigate the synchronization of activity rhythms to light and feeding in single goldfish given a single daily meal and exposed to different light regimes. In the 1st experiment, the group of fish fed at the end of the day showed a longer FAA (228 +/- 27 min) than the group fed 1 h after lights-on (97 +/- 31 min). In the 2nd experiment, goldfish under conflicting zeitgebers, LD (T = 26) and feeding (T = 24) cycles, initially synchronized to the light cycle although feeding time gradually assumed greater importance as the experiment progressed. In the 3rd experiment, after altering the feeding cycles by advancing or delaying feeding time by 4 h in LD 0.25:23.75, most goldfish resynchronized to the feeding stimulus and developed FAA. In conclusion, the authors' results revealed that FAA in goldfish is driven by an endogenous timing system that is entrainable by periodic feeding and influenced by the light regime.     

Single gene deletions of orexin, leptin, neuropeptide Y, and ghrelin do not appreciably alter food anticipatory activity in mice

Timing activity to match resource availability is a widely conserved ability in nature. Scheduled feeding of a limited amount of food induces increased activity prior to feeding time in animals as diverse as fish and rodents. Typically, food anticipatory activity (FAA) involves temporally restricting unlimited food access (RF) to several hours in the middle of the light cycle, which is a time of day when rodents are not normally active. We compared this model to calorie restriction (CR), giving the mice 60% of their normal daily calorie intake at the same time each day. Measurement of body temperature and home cage behaviors suggests that the RF and CR models are very similar but CR has the advantage of a clearly defined food intake and more stable mean body temperature. Using the CR model, we then attempted to verify the published result that orexin deletion diminishes food anticipatory activity (FAA) but observed little to no diminution in the response to CR and, surprisingly, that orexin KO mice are refractory to body weight loss on a CR diet. Next we tested the orexigenic neuropeptide Y (NPY) and ghrelin and the anorexigenic hormone, leptin, using mouse mutants. NPY deletion did not alter the behavior or physiological response to CR. Leptin deletion impaired FAA in terms of some activity measures, such as walking and rearing, but did not substantially diminish hanging behavior preceding feeding time, suggesting that leptin knockout mice do anticipate daily meal time but do not manifest the full spectrum of activities that typify FAA. Ghrelin knockout mice do not have impaired FAA on a CR diet. Collectively, these results suggest that the individual hormones and neuropepetides tested do not regulate FAA by acting individually but this does not rule out the possibility of their concerted action in mediating FAA.     

Daily cycles in plasma and ocular melatonin in demand-fed sea bass, Dicentrarchus labrax L.

Melatonin is regarded as an internal zeitgeber, involved in the synchronization to light of the daily and seasonal rhythms of vertebrates. To date, plasma and ocular melatonin in fish have been extensively surveyed almost solely in freshwater species – with the exception of some migrating species of salmonids. In the present paper, melatonin levels of a marine species (sea bass, Dicentrarchus labrax L) were examined. In addition, the daily rhythms of the demand-feeding activity of sea bass, a fish species characterized by a dual phasing capacity (i.e. the ability to switch between diurnal and nocturnal behaviour), were investigated before sampling. Sea bass, distributed in 12 groups of four fish and kept under constant water temperature and salinity, were exposed to a 12 h light:12 h dark cycle (200:0 lx, lights on at 0800 hours). After 4 weeks recording, the animals were killed at 0900, 1200, 1400, 1600, 1900, 2100, 2400, 0200, 0400, 0700 and 0900 hours. Actograms of demand-feeding records revealed a nocturnal feeding behaviour, with some cases of spontaneous inversions in phasing. Melatonin levels in plasma peaked in the middle of the dark phase, dropping after lights on. Melatonin in the eye, on the contrary, exhibited an inverse profile, with high levels during daytime and low levels at night. These results suggest that melatonin in the plasma and the eye may act independently on the flexible circadian system of sea bass. Accepted: 30 January 1997     

The effect of meal size and meal duration on food anticipatory activity in greenback flounder

Latency of food anticipatory activity (FAA) in greenback flounder Rhombosolea tapirina was about 21 days. Fish fed at meal sizes of 0·25 and 0·5% W day⁻¹ exhibited FAA under meal durations of 1, 3 and 7 h. Fish fed at 1·5% W day⁻¹ showed FAA only at a meal duration of 1 h. At each meal size, FAA was shorter and lower the longer the duration of the meal. The mean durations of FAA and post-feeding activity were correlated positively ( r =0·87; P <0·01; n =7). FAA persisted for <3 days during food deprivation. It is suggested that greenback flounder was capable of evaluating the energetic and temporal impacts of a single daily meal.     

Time Learning and Anticipatory Activity in Groups of Arctic Charr

Time learning and anticipatory activity were studied in five groups of 16–17 Arctic charr (Salvelinus alpinus) using self‐feeding devices with individual recognition (PIT (Passive Integrated Transponders)‐tags). The fish were kept under a LD 12:12 h cycle and periods of free access to the food were alternated with periods of time‐limited (2 h) access to food. Self‐feeding activity was significantly related to the light period in unrestricted conditions while related to the feeding periods during time‐limited access to food. The fish learned to concentrate their feeding activity to the restricted mealtime (>50% of the daily self‐feeding activity) within 10 d. The food anticipatory activity, measured as an increased self‐feeding activity before the feeding time and as aggressive interactions close to the trigger, was significant in both cases. The dominant individuals increased the trigger activity in advance of the time‐restricted reward period and subdominant individuals approached the trigger also in advance, inducing aggressive interactions. Thus, anticipating and learning a temporally predictable food source was pronounced in groups of self‐feeding Arctic charr.     

Food restricted schedules promote differential lipoperoxidative activity in rat hepatic subcellular fractions

Restricted access to food (from 12:00 to 14:00 h) produces a behavioral activation known as food anticipatory activity (FAA), which is a manifestation of the food entrained oscillator (FEO). Peripheral oscillators, especially in the liver, are thought to be part of the FEO. A variety of metabolic adaptations have been detected in the liver during the expression of this oscillator, including activation of mitochondrial respiration and changes in the cytoplasmic and mitochondrial redox states. Biological clocks are regulated by redox-sensitive factors. The present study explored the lipoperoxidative activity (LP) in the liver during the activity of the FEO. Conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS), with and without Fe2+-supplementation, were quantified in six subcellular fractions: whole homogenate, plasma membrane, mitochondria, microsomes, nucleus, and cytosol. The experimental protocol involved control groups of ad libitum fed and 24-h fasted rats, and groups under the restricted food schedule (RFS) which were sampled before FAA (08:00 h), during FAA (11:00 h) and after feeding (14:00 h). Clear differences in pro-oxidant activity was observed between ad libitum fed and 24-h fasted rats in almost all the subcellular fractions studied. RFS rats presented: CD levels more similar to the fasted rats, even at 14:00 h, after food presentation, and basal and Fe2+-supplemented TBARS levels tended to be lower than both controls, suggesting an increased antioxidant capacity associated with food restriction. In addition, a microarray analysis showed that several isoforms of peroxiredoxins, a family of antioxidant and hydrogen peroxide-catabolizing enzymes, were consistently up-regulated in each and every condition in which RFS was applied. Together, these data indicate a rheostatic adaptation of the liver in the handling of pro-oxidant reactions during the activity of the FEO.     

Ghrelin mediates anticipation to a palatable meal in rats

Food anticipatory activity (FAA) is displayed in rats when access to food is restricted to a specific time frame of their circadian phase, a behavior thought to reflect both hunger and the motivation to eat. Rats also display FAA in a feeding schedule with ad libitum access to normal chow, but limited availability of a palatable meal, which is thought to involve mainly motivational aspects. The orexigenic hormone ghrelin has been implicated in FAA in rodents with restricted access to chow. Because ghrelin plays an important role not only in the control of food intake, but also in reward, we sought to determine the role of ghrelin in anticipation to a palatable meal. Plasma ghrelin levels of non-restricted rats that anticipated chocolate correlated positively with FAA and were increased compared with chow-fed control rats. Furthermore, centrally injected ghrelin increased, whereas an antagonist of the ghrelin receptor decreased, the anticipation to chocolate. Therefore, we hypothesize that central ghrelin signaling is able to mediate the motivational drive to eat.     

Feeding time synchronizes clock gene rhythmic expression in brain and liver of goldfish (Carassius auratus)

Little is known about the feeding time dependence of clock gene expression in fish. The aim of the present study was to investigate whether a scheduled feeding time can entrain the rhythmic expression of several clock genes (period and cryptocrome) in the brain and liver of a teleost, the goldfish. Fish maintained under continuous light (LL) conditions were divided into 3 groups. Two groups were fed daily at 1000 h and 2200 h, respectively, and the third group was subjected to a random schedule regime. After 30 days, the fishes under 24-h food deprivation were sacrificed through a 24-h cycle, and clock gene expression in the optic tectum, hypothalamus, and liver was quantified by real-time PCR. The findings pointed to differences between the central and peripheral tissues studied. In the absence of a light-dark cycle (constant light), a scheduled feeding regime was necessary and sufficient to maintain both the rhythmic expression of several clock genes in the optic tectum and hypothalamus, as well as daily rhythms in locomotor activity. In contrast, neither locomotor activity nor clock gene expression in brain tissues was synchronized in randomly fed fish. However, in the liver, most of the clock genes studied presented significant daily rhythms in phase (related to the time of the last meal) in all 3 experimental groups, suggesting that the daily rhythm of clock genes in this organ only depends on the last meal time. The data suggest that, as in mammals, the smooth running of the food entrainable oscillator (FEO) in fish involves the rhythmic expression of several clock genes (Per1 and Cry3) in the central and peripheral structures. The results also indicate that the food anticipatory activity (FAA) in goldfish is not only the result of rhythmic clock gene expression in the liver because rhythmic clock gene expression was observed in randomly fed fishes, while FAA was not observed.     

Stress-induced effects on feeding behavior and growth performance of the sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>): a self-feeding approach

Repetitive aquaculture-related protocols may act as cyclic stressors that induce chronic stress in cultured fish. The sea bass is particularly sensitive to stressful conditions and the mere presence of humans will disturb feeding behavior. In this paper, we study whether chronic stress induced by repetition of acute stress protocols affects long-term feeding behavior and growth performance in sea bass and whether exogenous cortisol may induce stress-like changes in these parameters. We demonstrate that both chronic stress and dietary cortisol decrease food intake and have a negative effect on feed conversion efficiency, severely impairing sea bass performance. Both experimental approaches induced changes in the daily feeding activity by lengthening the active feeding periods. Fish subjected to a cyclic stressor modify their daily feeding pattern in an attempt to avoid interference with the time of the stressor. The delay in feeding when fish are acutely and repeatedly stressed could be of substantial adaptive importance.     

Effects of feeding low fishmeal diets with increasing soybean meal levels on growth,gut histology and plasma biochemistry of sea bass

The aquaculture industry depends upon the development of sustainable protein sources to replace fishmeal (FM) in aquafeeds and the products derived from soybeans are some of the most studied plant feedstuffs. A key area of investigation for continuing to improve modern aquafeeds includes the evaluation of varying proportions and combinations of plant ingredients to identify mixtures that are more efficiently utilized by the fish. This study investigated the effects of increasing soybean meal (SBM) by replacing a mix of plant ingredients in low FM (20%) diets on growth, blood biochemistry profile and gut histology on European sea bass. Five isonitrogenous and isolipidic experimental diets were formulated: four diets containing increasing SBM levels (0, 10, 20 and 30%; 0SBM, 10SBM, 20SBM and 30SBM, respectively) with a low content of FM (20%) and one control diet (0% SBM; 35% FM). Diets containing SBM brought to comparable performance and protein utilization, while 0SBM had negative impact on feed conversion rate and protein utilization. Blood parameters suggested an optimal nutritional status under all feeding treatments, even though slightly decreased values were reported at increasing dietary SBM. Histology examination did not show any changes indicative of soy-induced enteritis. We can conclude that for European sea bass: (i) different blends of plant protein did not affect feed intake despite the 20% FM dietary level; (ii) the inclusion of SBM maintains optimal growth and feed utilization in low FM diets; (iii) blood biochemistry profile showed a good nutritional status under all feeding regimes; (iv) no evidence of soy-induced enteritis was reported in any group fed low FM diets. For formulation of practical diets in on-growing of European sea bass, SBM up to 30% can be successfully incorporated into feeds containing low FM inclusion.     

Feeding entrainment of locomotor activity rhythms in the goldfish is mediated by a feeding-entrainable circadian oscillator

Periodic food availability can act as a potent zeitgeber capable of synchronizing many biological rhythms in fishes, including locomotor activity rhythms. In the present paper we investigated entrainment of locomotor rhythms to scheduled feeding under different light and feeding regimes. In experiment 1, fish were exposed to a 12:12?h light/dark cycle and fed one single daily meal in the middle of the light phase. In experiment 2, we tested the effect of random versus scheduled feeding on the daily distribution of activity. During random feeding, meals were randomly scheduled with intervals ranging from 12 to 36?h, while scheduled feeding consisted of one single daily meal set in the middle of the light or dark phase. Finally, in experiment 3, we studied the synchronization of activity rhythms to feeding under constant darkness (DD) and after shifting the feeding cycle by either advancing or delaying the feeding cycle by 9?h. The results revealed that goldfish synchronized to feeding, overcame light entrainment and significantly changed their daily distribution of activity according to their feeding schedule. In addition, the daily activity pattern modulated by feeding differed between layers: a peak of activity being noticeable directly after feeding at the bottom, while an anticipatory behaviour was obvious at the surface of the tank. Under DD and no food, free-running rhythms averaging 25.5?± 1.9?h (mean?±?SD) were detected. In conclusion, some properties of feeding entrainment (e.g. anticipation of the feeding time, free-running rhythms following termination of periodic feeding, and the stability of ø after shifting the feeding cycle) suggested that goldfish have (a) separate but tightly coupled light- and food-entrainable oscillators, or (b) a single oscillator that is entrainable by both light and food (one synchronizer being eventually stronger than the other).     

Unique food-entrained circadian rhythm in cysteine414-alanine mutant mCRY1 transgenic mice

Food availability is a potent environmental cue that directs circadian locomotor activity in rodents. Daily scheduled restricted feeding (RF), in which the food available time is restricted for several hours each day, elicits anticipatory activity. This food-anticipatory activity (FAA) is controlled by a food-entrainable oscillator (FEO) that is distinct from the suprachiasmatic nucleus (SCN), the master pacemaker in mammals. In an earlier report, we described generation of transgenic (Tg) mice ubiquitously overexpressing cysteine414-alanine mutant mCRY1. The Tg mice displayed long locomotor free-running periods (approximately 28 h) with rhythm splitting. Furthermore, their locomotor activity immediately re-adjusted to the advance of light–dark cycles (LD), suggesting some disorder in the coupling of SCN neurons. The present study examined the restricted feeding cycle (RF)-induced entrainment of locomotor activity in Tg mice in various light conditions. In LD, wild-type controls showed both FAA and LD-entrained activities. In Tg mice, almost all activity was eventually consolidated to a single bout before the feeding time. The result suggests a possibility that in Tg mice the feeding cycle dominates the LD cycle as an entrainment agent. In constant darkness (DD), wild-type mice exhibited robust free-run activity and FAA during RF. For Tg mice, only the rhythm entrained to RF was observed in DD. Furthermore, after returning to free feeding, the free-run started from the RF-entrained phase. These results suggest that the SCN of Tg mice is entrainable to RF and that the mCRY1 mutation alters the sensitivity of SCN to the cycle of nonphotic zeitgebers.

     

Trypsin activity and physiological aspects in larval rearing of European sea bass (Dicentrarchus labrax) using live prey and compound diets

The aim of this study was to evaluate the potential of a compound diet as a live prey substitute for feeding European sea bass larvae ( Dicentrarchus labrax L.). The effect of a commercial diet (Nippai ML feed) and live prey ( Artemia nauplii) on tryptic enzyme activity, protein content, growth (standard length) and survival rates of sea bass larvae were tested during a 27-day rearing experiment. Sea bass larvae were divided into two groups. The live food group (control group) was fed exclusively on newly hatched Artemia nauplii (Inve AF grade), the test group was fed exclusively with the compound diet from day 15 onwards. As trypsin has been demonstrated to be a useful indicator for evaluating digestibility of food and the nutritional condition of fish larvae, individual tryptic enzyme activity was determined in both feeding groups. Larvae older than 14 days after hatching and fed on live food showed a significantly higher tryptic enzyme activity than larvae fed the compound diet. A similar relationship between tryptic activity and standard length in both test groups was detected only in small larvae (standard length < 7 mm). The usefulness of proteolytic enzyme activity measurements in larval fish research, as well as its use in aquaculture nutrition research, was confirmed. Protein content, increase in length and survival rates of the sea bass larvae were additionally determined in order to evaluate an influence on the diet. The protein content of larvae fed the Artemia nauplii was higher and the growth of larvae fed the compound diet was reduced. Larval mortality was not affected by the diet given.     

Potential use of high levels of vegetal proteins in diets for market-sized gilthead sea bream (Sparus aurata)

The effect of partial or total dietary substitution of fishmeal (FM) by vegetal protein sources on growth and feed efficiency was carried out in on-growing gilthead sea bream (mean initial weight 131 g). The Control diet (FM 100) contained FM as the primary protein source, while in Diets FM 25 and FM 0 the FM protein was replaced at 75% and 100%, respectively, by a vegetable protein mixture consisting of wheat gluten, soybean meal, rapeseed meal and crystalline amino acids. Diets FM 25 and FM 0 also contained krill meal at 47 g/kg in order to improve palatability. At the end of the trial (after 158 d), fish survival was above 90%. Final weight and the specific growth rate were statistically lower in fish fed the Control diet (361 g and 0.64%/d), compared with 390–396 g and 0.69–0.70%/d after feeding vegetal diets. No significant differences were found regarding feed intake and feed conversion ratio. The digestibility of protein and amino acids (determined with chromium oxide as indicator) was similar in all diets. The blood parameters were not significantly affected by treatments. The activity of trypsin and pepsin was significantly reduced after feeding Diet FM 0. In the distal intestine, the villi length in fish fed Diet FM 25 was significantly longer and the intestine of the fish fed the FM 100 diet showed a smaller number of goblet cells. In conclusion, a total FM substitution by a vegetal mix supplemented with synthetic amino acids in on-growing sea bream is feasible.     

Photic and Pineal Modulation of Food Anticipatory Circadian Activity Rhythms in Rodents

Restricted daily feeding schedules entrain circadian oscillators that generate food anticipatory activity (FAA) rhythms in nocturnal rodents. The location of food-entrainable oscillators (FEOs) necessary for FAA remains uncertain. The most common procedure for inducing circadian FAA is to limit food access to a few hours in the middle of the light period, when activity levels are normally low. Although light at night suppresses activity (negative masking) in nocturnal rodents, it does not prevent the expression of daytime FAA. Nonetheless, light could reduce the duration or magnitude of FAA. If so, then neural or genetic ablations designed to identify components of the food-entrainable circadian system could alter the expression of FAA by affecting behavioral responses to light. To assess the plausibility of light as a potential mediating variable in studies of FAA mechanisms, we quantified FAA in rats and mice alternately maintained in a standard full photoperiod (12h of light/day) and in a skeleton photoperiod (two 60 min light pulses simulating dawn and dusk). In both species, FAA was significantly and reversibly enhanced in the skeleton photoperiod compared to the full photoperiod. In a third experiment, FAA was found to be significantly attenuated in rats by pinealectomy, a procedure that has been reported to enhance some effects of light on behavioral circadian rhythms. These results indicate that procedures affecting behavioral responses to light can significantly alter the magnitude of food anticipatory rhythms in rodents.     

Differential effects of meal size and food energy density on feeding entrainment in goldfish

The synchronizing stimulus, its transduction site, and the afferent pathways responsible for feeding entrainment remain unknown. In fish, the role of the diet in the development of feeding anticipatory activity (FAA) is not well understood and fundamental questions on the mechanisms of feeding entrainment, such as the meal characteristics required to develop FAA, remain unexplored. To test the entraining properties of daily meals with different sizes and energy densities, activity rhythms were studied after a 12-h shift of the feeding cycle in individual goldfish under constant light. In the 1st experiment, the energy content of a control diet (16.7 kJ/g) was diluted by replacing 50% (8.3 kJ/g) or 90% (1.7 kJ/g) of the diet with cellulose. However, the number of days required to stabilize FAA after the shift did not differ statistically between diets. In the 2nd experiment, meal size was modified by reducing the daily feeding ration to 0.5% and 0.1% b.wt.d(-1). In this case, differences in the entraining properties of the two feeding rations appeared because goldfish fed at 0.1% b.wt.d(-1) resynchronized faster than those fed at 0.5% b.wt.d(-1). These results revealed that the dilution of the dietary energy up to 1.7 kJ/g had no significant effect on the entraining properties of the feeding-entrainable oscillator (FEO), whereas the reduction of the meal size to 0.1% b.wt.d(-1) provoked a faster resynchronization after shifting the daily meal cycle. Taken together, these results suggest that gut distension may be involved in feeding entrainment, as a reduction in meal size but not in the amount of dietary energy supplied significantly shortened the time required for resynchronization and highlighted the different synchronizing properties of meal size and energy density as zeitgebers for the FEO.     

Scheduled feeding alters the timing of the suprachiasmatic nucleus circadian clock in dexras1-deficient mice

Restricted feeding (RF) schedules are potent zeitgebers capable of entraining metabolic and hormonal rhythms in peripheral oscillators in anticipation of food. Behaviorally, this manifests in the form of food anticipatory activity (FAA) in the hours preceding food availability. Circadian rhythms of FAA are thought to be controlled by a food-entrainable oscillator (FEO) outside of the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. Although evidence suggests that the FEO and the SCN are capable of interacting functionally under RF conditions, the genetic basis of these interactions remains to be defined. In this study, using dexras1-deficient (dexras1(-/-)) mice, the authors examined whether Dexras1, a modulator of multiple inputs to the SCN, plays a role in regulating the effects of RF on activity rhythms and gene expression in the SCN. Daytime RF under 12L:12D or constant darkness (DD) resulted in potentiated (but less stable) FAA expression in dexras1(-/-) mice compared with wild-type (WT) controls. Under these conditions, the magnitude and phase of the SCN-driven activity component were greatly perturbed in the mutants. Restoration to ad libitum (AL) feeding revealed a stable phase displacement of the SCN-driven activity component of dexras1(-/-) mice by ～2?h in advance of the expected time. RF in the late night/early morning induced a long-lasting increase in the period of the SCN-driven activity component in the mutants but not the WT. At the molecular level, daytime RF advanced the rhythm of PER1, PER2, and pERK expression in the mutant SCN without having any effect in the WT. Collectively, these results indicate that the absence of Dexras1 sensitizes the SCN to perturbations resulting from restricted feeding. (Author correspondence: haiying.cheng@utoronto.ca ).     

Behavioral and neurochemical investigation of circadian time-place learning in the rat

The ability to form an association between the time and the place of food availability, namely time-place learning, is presumably important for survival. The present study was designed to examine time-place learning and to identify exogenous and endogenous factors that may affect this behavior in rats. In an initial experiment, rats displayed poor time-place behavior and appeared to prefer the feeder that was closer to the center aisle and water supply. When these cues were minimized in a subsequent experiment, rats consistently displayed the time-place discrimination by exhibiting food anticipatory activity (FAA) at the correct location prior to each meal time. These rats also showed significant correlations between the level of FAA and the amount of dopamine turnover (the dihydroxyphenylacetic acid/dopamine ratio) in the nucleus accumbens and paraventricular nucleus of the hypothalamus, indicating possible involvement of regional dopaminergic activity in time-place behavior. No correlation was found for norepinephrine, epinephrine, or serotonin. In addition, the correlation between FAA and dopamine turnover was not found when rats were entrained to only one meal per day. Together, the data suggest that rats can learn the time-place discrimination under proper experimental conditions and that dopamine may play a role in the expression of this behavior.     

Diurnal changes in blood metabolites and their relation to plasma growth hormone and time of feeding in mithun heifers (Bos frontalis)

The aim of the present study was to investigate what, if any, diurnal changes occur in blood metabolites in relation to plasma growth hormone (GH) and feeding time among mithun (Bos frontalis), a semi-wild ruminant. Blood samples were collected at hourly intervals during a 24 h span from 6 mithun heifers (averaging 2.5 yr of age and averaging 230 kg in weight) that were fed twice a day at 11:00 and 16:00 h. Samples were assayed for plasma GH and blood metabolites, non-esterified fatty acids (NEFA), glucose, and alpha-amino nitrogen. The total sampling period was divided into a 1) postprandial (after meal) period (period I: 11:00 to 21:00 h) and 2) interprandial period (period II: 22:00 to 10:00 h) and also into night (20:00 to 05:00 h) and day (06:00 to 10:00 h) periods for statistical analysis. Plasma glucose and alpha-amino nitrogen levels increased (p<0.01), and plasma NEFA and GH decreased (p<0.01) after each meal. No diurnal rhythmicity was detected in plasma glucose or alpha-amino nitrogen levels. Interestingly, plasma NEFA and GH levels were higher (p<0.01) during the interprandial (period II) and night periods, indicating an energy deficit that occurred progressively during the interprandial period of nocturnal feed deprivation. In twice-daily-fed mithuns we conclude that: 1) plasma metabolites and GH exhibited a definite pattern of change with time of feeding; 2) concentrations of plasma NEFA were higher nocturnally due to an energy deficit and that GH levels were higher during the interprandial period after the second meal; 3) the interprandial period after the second feeding may be considered to constitute a short-term food deprivation; 4) the longer interprandial period of 19 h in this study between the second and subsequent morning meal may be changed into equally divided feedings to minimize the short-term energy deficit; and 5) blood sampling for blood metabolites in mithuns should be conducted at a fixed time of day with special emphasis on time of feeding.