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Influence of time of food administration on daily rhythm of total locomotor activity in ponies
Journal of Veterinary Behavior (2013) 8, 40-45
RESEARCH
Influence of time of food administration on daily rhythm of total locomotor activity in ponies Giuseppe Piccione, Claudia Giannetto, Simona Marafioti, Michele Panzera, Anna Assenza, Francesco Fazio Laboratory of Veterinary Chronophysiology, Department of Experimental Science and Applied Biotechnology, University of Messina, Messina, Italy. KEYWORDS: circadian rhythms; feeding schedules; locomotor activity; horse
Abstract The activity rhythm can be entrained by nonphotic cues, especially food availability. Daily feeding schedules can act as ‘‘Zeitgeber’’ to synchronize the circadian system. In this study, the authors evaluated the influence of different feeding schedules on the daily rhythm of total locomotor activity in ponies. Six clinically healthy Shetland ponies were kept under natural 12/12-h light/dark cycle and monitored during 4 different feeding schedules: fed twice (at 8 AM and 8 PM), fasted, fed at 8 PM, fed at 8 AM, and fed twice a day again. Water was available ad libitum. To record total activity, we equipped the animals with actigraphy-based data loggers (Actiwatch-Mini). Two-way analysis of variance revealed a significant main effect of the time of day and no effect of feeding schedules on the amount of total locomotor activity. Locomotor activity showed daily rhythmicity in all experimental conditions, with diurnal acrophase. The amplitude of the rhythms and their robustness statistically changed across all experimental conditions. Our findings support the theory that when given essentially free choice hay, the time of feeding did not entrain activity, and suggest that feeding behavior can influence the distribution of locomotor activity during a 24-hour period in ponies, with changes in the amplitude and robustness of the rhythm. Ó 2013 Elsevier Inc. All rights reserved.
Introduction The adaptation to environmental changes brought about by the succession of day and night offers strong advantages for the efficient use of natural resources and, hence, is a major evolutionary selection criterion (Pittendrigh, 1993). Not surprisingly, the simplest species living on earth have evolved timekeeping mechanisms that enable them to
Address for reprint requests and correspondence: Giuseppe Piccione, Laboratory of Veterinary Chronophysiology, Department of Experimental Science and Applied Biotechnology, University of Messina, 98168 Messina, Italy; Tel: 139-0903503584; Fax: 139-0903503975. E-mail: [email protected] 1558-7878/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. doi:10.1016/j.jveb.2012.02.003
predict and adapt to upcoming time-dependent events (Harmer et al., 2001). Daily variations in behavior and physiology are controlled by a circadian timing system consisting of a network of oscillatory structures (Challet, 2010). Circadian clocks anticipate daily events and adapt behavior and physiology in an attempt to minimize energy expenditure and maximize the chance of survival. In mammals, a master clock, located in the suprachiasmatic nuclei (SCN) of the hypothalamus, adjusts the timing of other selfsustained oscillators in the brain and peripheral organs (Challet, 2010). For most species, daily light–dark (L/D) cycles are the primary environmental stimulus (Zeitgeber) for the entrainment of the SCN pacemaker. The SCN receives light information from the retina and regulates
Piccione et al
Daily rhythm of total locomotor activity in ponies
diverse physiological processes by synchronizing molecular clockwork mechanisms consisted by a core group of clock genes in each cell (Murphy et al., 2009). The existence of another clockdthe food-entrainable clock (FEC)dhas been demonstrated (Feillet et al., 2006). The FEC is believed to be anatomically and functionally distinct from the light-entrainable oscillator (SCN) (Edmonds and Adler, 1977a,b; Stephan, 1989; Stephan and Becker, 1989). This clock is suggested to have multiple outputs, including locomotor activity, temperature, and hormonal and metabolic signals (Feillet et al., 2006). The 2 systems would have to be strongly coupled so that one would entrain the other. Although the FEC system can function in the absence of the SCN, the SCN may be required to maintain the amplitude of meal-entrained rhythms and may control the period and phase of FEC as long as food availability is unrestricted. It is suggested that the coupling between the FEC and the light-entrainable pacemaker (LEP) on the SCN is asymmetrical, as FEC is usually coupled to LEP but has a weak effect on it (Angle`s-Pujolra`s et al., 2006). Food availability may affect the circadian organization of daily rhythms. It is not clear how feeding activity affects the circadian pacemaker. The major parameter affected by food availability is the rhythm of daily locomotor activity. Many studies were conduced in laboratory animals (Boulos et al., 1980; Clarke and Coleman, 1986; De Groot and Rusak, 2004) and sheep (Piccione et al., 2007) to investigate the influence of food availability on the daily rhythm of locomotor activity. In all these studies, the animals were subjected to a restricted access to food during the day. This study investigates the influence of the timing of food availability on daily total locomotor activity and the temporal characteristics of locomotor activity rhythms in ponies.
Material and methods In our study, 6 clinically healthy ponies (Shetland pony breed, gelding, 8-10 years old, mean body weight: 150-180 kg) in good nutritional condition were used. Animals were housed individually in a 12-m2 soundproof (panels of rigid fibreglass) light-tight box equipped with an opening window and bedding of wood shavings. The visual and acoustic isolation of each animal from the other avoided the social entrainment of the circadian behavioral rhythms (Davidson and Menaker, 2003). The animals were put in the experimental box 30 days before starting the study to avoid changes in the behavior and physiology of animals caused by the state of fear induced by isolation (Bagshaw et al., 1994). Thermal and hygrometric records were collected inside the box for the whole study using a data logger (Gemini, West Sussex, United Kingdom). Minimum temperature during the experimental period was 15.5 C, the maximum was 18.5 C, and the range of mean humidity was 55%-60%. Animals were kept under natural (February)
41
12:12-h L/D cycle (6 AM at sunrise, 6 PM at sunset), in Messina, Italy (latitude: 38 , 110 , 4300 ; longitude: 15 , 260 , 100 ), at 150 m above sea level. For the first 4 days, animals were fed twice (8 AM and 8 PM); during the fifth and sixth days, animals were fasted; from day 7 to day 10, animals were fed once (8 PM); from day 11 to day 14, animals were fed once (8 AM); and from day 15 to day 18, animals were fed twice again. During all feeding schedules, ponies received 4 kg hay/d. Before subsequent feeding, 0.8%-1.5% of the hay offered was removed. This condition suggests that ponies self-regulated their intake over the 24 hours. No concentrates were administered, and the nutrients needed were provided in adequate amounts by the forage. Water was available ad libitum (Figure 1). The total activity of the ponies, which included different behaviors, such as feeding, drinking, walking, and grooming, as well as all conscious and unconscious movements, was measured after they had been on a particular schedule for at least 2 days (day 4 [twice], 6 [fasting], 10 [once at 8 PM], 14 [once at 8 AM], and 18 [twice]). To record activity, we equipped the animals with Actiwatch-Mini (Cambridge Neurotechnology Ltd., Cambridge, United Kingdom), an actigraphy-based data logger that records a digitally integrated measure of motor activity. Actigraphs were placed using headstalls that were accepted without any obvious disturbance; previous investigations (Bertolucci et al., 2008) have shown that collars correctly identified the behavior parameters of activity and feeding. It is important to stress that owing to this improved way of recording activity data, there is no need for sensitivity setting, as the Actiwatch unit records all movements greater than 0.05 gravitational acceleration. Activity was monitored with a sampling interval of 5 minutes. The total daily amount of activity and the amount of activity during the photophase and the scotophase were calculated using Actiwatch Activity Analysis 5.06 (Cambridge Neurotechnology Ltd) and expressed in arbitrary units. Two-way analysis of variance (ANOVA) was applied to determine statistical difference caused by feeding schedules and photophase and scotophase. Two-way repeated measures ANOVA was used to determine significant differences caused by the time of day and feeding schedules (P , 0.05 was considered statistically significant). Data were analyzed using the software STATISTICA 9.1 (StatSoft Inc., Tulsa, OK). Using cosinor rhythmometry (Nelson et al., 1979), 4 rhythmic parameters were determined, including mesor (mean level), amplitude (half the range of oscillation), acrophase (time of peak), and robustness (strength of rhythmicity). Rhythm robustness (stationarity of a rhythm) was computed as the quotient of the variance associated with sinusoidal rhythmicity and the total variance of the time series (Refinetti, 2004). Robustness .14% is higher than the noise level and indicates statistically significant rhythmicity. One-way ANOVA was applied to determine statistically significant differences in the rhythmic parameters among
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Journal of Veterinary Behavior, Vol 8, No 1, January/February 2013
Figure 1 Summary of experimental conditions. White and black bars indicate photophase and scotophase. *Food administration. YDay of total locomotor activity recording.
the feeding schedules. All housing and care conformed to the standards recommended by the Guide for the Care and Use of Laboratory Animals and Directive 86/609 CEE.
Results Visual inspection of actograms showed that total locomotor activity was not evenly distributed throughout the day, but was mainly diurnal, except during the fasting period. Figure 2 shows a representative actogram of the total locomotor activity recorded in a pony during the 5 experimental periods. Statistical significant difference in the amount of activity between photophase and scotophase (P , 0.0001; F(4,50) 5 31.20) was observed in all experimental periods.
Higher levels of activity were observed during the photophase (Figure 2). There was no difference in the amount of activity recorded within each photophase across all feeding schedules. The same condition was observed for the amount of activity recorded within each scotophase. Twoway ANOVA revealed a significant (F(24,600) 5 2.46; P , 0.0001) main effect of the time of day and no effect of feeding schedules on the amount of total locomotor activity. Ponies spent about 13 h/d to completely consume 4 kg of hay when provided once a day, and about 6 hours to completely consume of 2 kg of hay when provided twice. The application of the periodic model and statistical analysis of the cosinor enabled us to define the periodic parameters. Mesor values and acrophase did not change among the experimental periods. Acrophase was always
Figure 2 Actograms of total locomotor activity record in a pony subjected to a 12:12-h light/dark cycle and different feeding schedules. White and black bars indicate photophase and scotophase.
Piccione et al
Daily rhythm of total locomotor activity in ponies
Figure 3 Mean 6 standard error of mean (SEM) of acrophases observed during the different experimental periods. White and black bars indicate photophase and scotophase.
diurnal (Figure 3). The amplitude of the rhythm statistically changed among the experimental periods (P , 0.002) and was statistically lower (P , 0.05) during the fasting period than the other ones (Figure 4). Robustness was influenced by experimental periods (P , 0.0002): it was higher when fed once a day at 8 PM and when fed once a day at 8 AM than the other ones (Figure 4).
Discussion Our results showed that the amount of daily total locomotor activity in ponies is not influenced by the time of food administration. In accordance with a previous study in which locomotor activity peaked in the middle of the light phase in trained and untrained horses (Piccione et al., 2011), higher levels of activity were observed in all experimental periods during the photophase in our study, and there were no differences between the amount of activity recorded within each photophase and scotophase across all feeding schedules. Although animals always received the same amount of ration (4 kg/d) that meets the absolute amount of nutrition daily and simulates an ad libitum administration, the different feeding schedules induced a different distribution of activity between all feeding schedules. In particular, during the fasting period, it was spread through the day compared with the other periods (Figure 2). In most cases, feeding schedules appear to entrain only a component of activity, which anticipates the daily feeding time. When food availability is limited to a several-hour interval at a particular time each day, mammals quickly develop a new component of daily behavioral activityda second period of arousal and increased locomotor activity that occurs shortly before the time of daily food presentation (Mistlberger, 1994). This component, known as food anticipatory activity (FAA), exhibits fundamental properties of a clock-controlled process, including resistance to entrainment and persistence in the absence of the Zeitgeber (Mistlberger, 1994). It is thought that FAA
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represents an evolutionary adaptation providing mammals with a highly flexible and efficient food-seeking program, one that is able, if necessary, to take advantage of food sources available at unusual times with respect to the animal’s typical daily rest–activity cycle (Storch and Weitz, 2009). In our study, FAA failed to emerge in all days tested. Replenishing food hoppers at the same time each day without caloric restriction does not induce food anticipation (Mistlberger, 2009). Ponies self-regulated their intake over the 24 hours. The physical form in which a feed is presented alters the rate of consumption and total dry matter intakes by horses and ponies. Both foraging and eating behavior are modified to allow optimal rates of food ingestion (Arnold, 1985). Horses distribute their foraging and feeding behavior across day and night (Martin et al., 2010). Their digestive system is designed for a small intake at frequent intervals. In the study by Ralston (1984), the horses were given free access to feed, and although confined to stalls, they engaged in meal patterns that were similar to those described under free ranging conditions. Stable conditions can influence the locomotor activity intensity, but not the feeding behavior (Piccione et al., 2008). Food has been considered a dominant Zeitgeber for peripheral clocks in various mammalian species (Damiola et al., 2000). Changes in the circadian pattern of eating and time spent eating are the behavioral response of the animal to the amount of food availability. The daily routine of maintenance behavior is determined by the timing of feeding, with other activities accommodated to it (Arnold, 1985). The overall rhythmic pattern of mares maintained outdoors in a large 4-acre pasture was both strongly ultradian and weakly circadian (Martin et al., 2010). In particular, the different time of food administration influenced the amplitude and the robustness of the rhythm. The absence of feeding behavior during the fasting period determined a statistical decrease of the amplitude of the daily rhythm of locomotor activity. The amplitude of the rhythms can be affected by many factors downstream from the oscillator responsible for timing the rhythms. If phase, precision, and period are unaltered, a change in amplitude is probably an effect downstream from the clock (Mistlberger, 2009). A more robust rhythm was observed during the period when the animals were fed once a day, and providing hay at 8 PM did not invert the feeding behavior. In the period when the animals were fed once at 8 PM, we might have expected an increase of activity during the scotophase after providing the food, whereas the animals maintained their feeding behavior and a diurnal activity. In mammals, it appears that the unresponsiveness of the SCN clock to feeding time reflects an inherent property of the central pacemaker, rather than a dominance of photic entrainment over food entrainment (Damiola et al., 2000). In the presence of a daily L/D cycle, the phase of the SCN is altered relatively little or not at all by daily feeding schedules, regardless of mealtime (Damiola et al., 2000; Stokkan et al., 2001).
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Journal of Veterinary Behavior, Vol 8, No 1, January/February 2013
Figure 4 Mean 6 standard error of mean (SEM) of the robustness and amplitude of the rhythm observed during the different experimental periods. *P , 0.05 versus other periods.
Ponies can distribute their activity in different components, without significantly modifying daily total activity according to the response to external stimuli, such as food availability.
Conclusion Our findings support the theory that when given essentially free choice hay, the time of feeding did not entrain activity, underlining a dominance of photic entrainment over food entrainment. Therefore, feeding behavior can influence the distribution of locomotor activity during a 24-hour period in ponies, with changes in the amplitude and robustness of the rhythm. These findings underline that care must always be taken in considering that feeding schedules must be adequate to ensure the physiological rhythm of the animals.
References Angle`s-Pujolra`s, M., Chiesa, J.J., Dı`ez-Noguera, A., Cambras, T., 2006. Motor activity rhythms of forced desynchronized rats subjected to restricting feeding. Physiol. Behav. 88, 30-38. Arnold, G.W., 1985. Ingestive behaviour. In: Fraser, A.F. (Ed.), World Animal Science A. Basic Information. Ethology of Farm Animals. A Comprehensive Study of the Behavioural Features of the Common Farm Animals. Elsevier, Amsterdam, The Netherlands, Oxford, UK, New York, NY, Tokyo, Japan, pp. 183-200. Bagshaw, C.S., Ralston, S.L., Fisher, H., 1994. Behavioral and physiological effect of orally administered tryptophan on horses subjected to acute isolation stress. Appl. Anim. Behav. Sci. 40, 1-12. Bertolucci, C., Giannetto, C., Fazio, F., Piccione, G., 2008. Seasonal variations in daily rhythms of activity in athletic horses. Animal 2, 10551060. Boulos, Z., Rosenwasser, A.M., Terman, M., 1980. Feeding schedules and circadian organization of behaviour in the rat. Behav. Brain Res. 1, 39-65.
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Challet, E., 2010. Interactions between light, mealtime and calorie restriction to control daily timing in mammals. J. Comp. Physiol. B 180, 631-644. Clarke, J.D., Coleman, G.J., 1986. Persistent meal-associated rhythms in SCN-lesioned rats. Physiol. Behav. 36, 105-113. Damiola, F., Le Minh, N., Preitner, N., Kornmann, B., Fleury-Olela, F., Schibler, U., 2000. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Genes Dev. 14, 2950-2961. Davidson, A.J., Menaker, M., 2003. Birds of a feather clock togethersometimes: social synchronization of circadian rhythms. Curr. Opin. Neurobiol. 13, 765-769. De Groot, M.H., Rusak, B., 2004. Housing conditions influence the expression of food-anticipatory activity in mice. Physiol. Behav. 83, 447-457. Edmonds, S.C., Adler, N.T., 1977a. Food and light as entrainers of circadian running activity in the rat. Physiol. Behav. 18, 915-917. Edmonds, S.C., Adler, N.T., 1977b. The multiplicity of biological oscillators in the control of circadian running activity in the rat. Physiol. Behav. 18, 921-930. Feillet, C.A., Albrecht, U., Etienne, C., 2006. ‘‘Feeding time’’ for the brain: a matter of clocks. J. Physiol. (Paris) 100, 252-260. Harmer, S.L., Panda, S., Kay, S.A., 2001. Molecular bases of circadian rhythms. Annu. Rev. Cell Dev. Biol. 17, 215-253. Martin, A.M., Elliot, J.A., Duffy, P., Blake, C.M., Attia, S.B., Katzs, L.M., Browne, J.A., Gath, V., McGivney, B.A., Hill, E.W., Murphy, B.A., 2010. Circadian regulation of locomotor activity and skeletal muscle gene expression in the horse. J. Appl. Physiol. 109, 1328-1336. Mistlberger, R.E., 1994. Circadian food-anticipatory activity: formal models and physiological mechanism. Neurosci. Biobehav. Rev. 18, 171-195. Mistlberger, R.E., 2009. Food-anticipatory circadian rhythms: concepts and methods. Eur. J. Neurosci. 30, 1718-1729.
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Murphy, B.A., Martin, A.M., Elliott, J.A., 2009. Light: dark, circadian, and ultradian regulation of motor activity and skeletal muscle gene expression in the horse. J. Equine Vet. Sci. 29, 313-314. Nelson, W., Tong, U., Lee, J., Halberg, F., 1979. Methods for cosinor rhythmometry. Chronobiologia 6, 305-323. Piccione, G., Bertolucci, C., Caola, G., Foa`, A., 2007. Effects of restricted feeding on circadian activity rhythms of sheep-a brief report. Appl. Anim. Behav. Sci. 107, 233-238. Piccione, G., Costa, A., Giannetto, C., Caola, G., 2008. Daily rhythms of activity in horses housed in different stabling conditions. Biol. Rhythm Res. 39, 79-84. Piccione, G., Giannetto, C., Marafioti, S., Casella, S., Fazio, F., Caola, G., 2011. Daily rhythms of rectal temperature and total locomotor activity in trained and untrained horses. J. Vet. Behav.: Clin. Appl. Res. 6, 115-120. Pittendrigh, C.S., 1993. Temporal organization: reflections of a Darwinian clock-watcher. Annu. Rev. Physiol. 55, 16-54. Ralston, S.L., 1984. Control of feeding in horses. J. Anim. Sci. 59, 1354-1361. Refinetti, R., 2004. Non-stationary time series and the robustness of circadian rhythms. J. Theor. Biol. 227, 571-581. Stephan, F.K., 1989. Forced dissociation of activity entrained to T cycles of food access in rats with suprachiasmatic lesions. J. Biol. Rhythms 4, 467-479. Stephan, F.K., Becker, G., 1989. Entrainment of anticipatory activity to various durations of food access. J. Biol. Rhythms 46, 731-741. Stokkann, K.A., Yamazaki, S., Tei, H., Sakaki, Y., Menaker, M., 2001. Entrainment of the circadian clock in the liver by feeding. Science 291, 490-493. Storch, K.F., Weitz, C.J., 2009. Daily rhythms of food-anticipatory behavioural activity do not require the known circadian clock. Proc. Natl. Acad. Sci. 106, 6808-6813.
RESEARCH
Influence of time of food administration on daily rhythm of total locomotor activity in ponies Giuseppe Piccione, Claudia Giannetto, Simona Marafioti, Michele Panzera, Anna Assenza, Francesco Fazio Laboratory of Veterinary Chronophysiology, Department of Experimental Science and Applied Biotechnology, University of Messina, Messina, Italy. KEYWORDS: circadian rhythms; feeding schedules; locomotor activity; horse
Abstract The activity rhythm can be entrained by nonphotic cues, especially food availability. Daily feeding schedules can act as ‘‘Zeitgeber’’ to synchronize the circadian system. In this study, the authors evaluated the influence of different feeding schedules on the daily rhythm of total locomotor activity in ponies. Six clinically healthy Shetland ponies were kept under natural 12/12-h light/dark cycle and monitored during 4 different feeding schedules: fed twice (at 8 AM and 8 PM), fasted, fed at 8 PM, fed at 8 AM, and fed twice a day again. Water was available ad libitum. To record total activity, we equipped the animals with actigraphy-based data loggers (Actiwatch-Mini). Two-way analysis of variance revealed a significant main effect of the time of day and no effect of feeding schedules on the amount of total locomotor activity. Locomotor activity showed daily rhythmicity in all experimental conditions, with diurnal acrophase. The amplitude of the rhythms and their robustness statistically changed across all experimental conditions. Our findings support the theory that when given essentially free choice hay, the time of feeding did not entrain activity, and suggest that feeding behavior can influence the distribution of locomotor activity during a 24-hour period in ponies, with changes in the amplitude and robustness of the rhythm. Ó 2013 Elsevier Inc. All rights reserved.
Introduction The adaptation to environmental changes brought about by the succession of day and night offers strong advantages for the efficient use of natural resources and, hence, is a major evolutionary selection criterion (Pittendrigh, 1993). Not surprisingly, the simplest species living on earth have evolved timekeeping mechanisms that enable them to
Address for reprint requests and correspondence: Giuseppe Piccione, Laboratory of Veterinary Chronophysiology, Department of Experimental Science and Applied Biotechnology, University of Messina, 98168 Messina, Italy; Tel: 139-0903503584; Fax: 139-0903503975. E-mail: [email protected] 1558-7878/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. doi:10.1016/j.jveb.2012.02.003
predict and adapt to upcoming time-dependent events (Harmer et al., 2001). Daily variations in behavior and physiology are controlled by a circadian timing system consisting of a network of oscillatory structures (Challet, 2010). Circadian clocks anticipate daily events and adapt behavior and physiology in an attempt to minimize energy expenditure and maximize the chance of survival. In mammals, a master clock, located in the suprachiasmatic nuclei (SCN) of the hypothalamus, adjusts the timing of other selfsustained oscillators in the brain and peripheral organs (Challet, 2010). For most species, daily light–dark (L/D) cycles are the primary environmental stimulus (Zeitgeber) for the entrainment of the SCN pacemaker. The SCN receives light information from the retina and regulates
Piccione et al
Daily rhythm of total locomotor activity in ponies
diverse physiological processes by synchronizing molecular clockwork mechanisms consisted by a core group of clock genes in each cell (Murphy et al., 2009). The existence of another clockdthe food-entrainable clock (FEC)dhas been demonstrated (Feillet et al., 2006). The FEC is believed to be anatomically and functionally distinct from the light-entrainable oscillator (SCN) (Edmonds and Adler, 1977a,b; Stephan, 1989; Stephan and Becker, 1989). This clock is suggested to have multiple outputs, including locomotor activity, temperature, and hormonal and metabolic signals (Feillet et al., 2006). The 2 systems would have to be strongly coupled so that one would entrain the other. Although the FEC system can function in the absence of the SCN, the SCN may be required to maintain the amplitude of meal-entrained rhythms and may control the period and phase of FEC as long as food availability is unrestricted. It is suggested that the coupling between the FEC and the light-entrainable pacemaker (LEP) on the SCN is asymmetrical, as FEC is usually coupled to LEP but has a weak effect on it (Angle`s-Pujolra`s et al., 2006). Food availability may affect the circadian organization of daily rhythms. It is not clear how feeding activity affects the circadian pacemaker. The major parameter affected by food availability is the rhythm of daily locomotor activity. Many studies were conduced in laboratory animals (Boulos et al., 1980; Clarke and Coleman, 1986; De Groot and Rusak, 2004) and sheep (Piccione et al., 2007) to investigate the influence of food availability on the daily rhythm of locomotor activity. In all these studies, the animals were subjected to a restricted access to food during the day. This study investigates the influence of the timing of food availability on daily total locomotor activity and the temporal characteristics of locomotor activity rhythms in ponies.
Material and methods In our study, 6 clinically healthy ponies (Shetland pony breed, gelding, 8-10 years old, mean body weight: 150-180 kg) in good nutritional condition were used. Animals were housed individually in a 12-m2 soundproof (panels of rigid fibreglass) light-tight box equipped with an opening window and bedding of wood shavings. The visual and acoustic isolation of each animal from the other avoided the social entrainment of the circadian behavioral rhythms (Davidson and Menaker, 2003). The animals were put in the experimental box 30 days before starting the study to avoid changes in the behavior and physiology of animals caused by the state of fear induced by isolation (Bagshaw et al., 1994). Thermal and hygrometric records were collected inside the box for the whole study using a data logger (Gemini, West Sussex, United Kingdom). Minimum temperature during the experimental period was 15.5 C, the maximum was 18.5 C, and the range of mean humidity was 55%-60%. Animals were kept under natural (February)
41
12:12-h L/D cycle (6 AM at sunrise, 6 PM at sunset), in Messina, Italy (latitude: 38 , 110 , 4300 ; longitude: 15 , 260 , 100 ), at 150 m above sea level. For the first 4 days, animals were fed twice (8 AM and 8 PM); during the fifth and sixth days, animals were fasted; from day 7 to day 10, animals were fed once (8 PM); from day 11 to day 14, animals were fed once (8 AM); and from day 15 to day 18, animals were fed twice again. During all feeding schedules, ponies received 4 kg hay/d. Before subsequent feeding, 0.8%-1.5% of the hay offered was removed. This condition suggests that ponies self-regulated their intake over the 24 hours. No concentrates were administered, and the nutrients needed were provided in adequate amounts by the forage. Water was available ad libitum (Figure 1). The total activity of the ponies, which included different behaviors, such as feeding, drinking, walking, and grooming, as well as all conscious and unconscious movements, was measured after they had been on a particular schedule for at least 2 days (day 4 [twice], 6 [fasting], 10 [once at 8 PM], 14 [once at 8 AM], and 18 [twice]). To record activity, we equipped the animals with Actiwatch-Mini (Cambridge Neurotechnology Ltd., Cambridge, United Kingdom), an actigraphy-based data logger that records a digitally integrated measure of motor activity. Actigraphs were placed using headstalls that were accepted without any obvious disturbance; previous investigations (Bertolucci et al., 2008) have shown that collars correctly identified the behavior parameters of activity and feeding. It is important to stress that owing to this improved way of recording activity data, there is no need for sensitivity setting, as the Actiwatch unit records all movements greater than 0.05 gravitational acceleration. Activity was monitored with a sampling interval of 5 minutes. The total daily amount of activity and the amount of activity during the photophase and the scotophase were calculated using Actiwatch Activity Analysis 5.06 (Cambridge Neurotechnology Ltd) and expressed in arbitrary units. Two-way analysis of variance (ANOVA) was applied to determine statistical difference caused by feeding schedules and photophase and scotophase. Two-way repeated measures ANOVA was used to determine significant differences caused by the time of day and feeding schedules (P , 0.05 was considered statistically significant). Data were analyzed using the software STATISTICA 9.1 (StatSoft Inc., Tulsa, OK). Using cosinor rhythmometry (Nelson et al., 1979), 4 rhythmic parameters were determined, including mesor (mean level), amplitude (half the range of oscillation), acrophase (time of peak), and robustness (strength of rhythmicity). Rhythm robustness (stationarity of a rhythm) was computed as the quotient of the variance associated with sinusoidal rhythmicity and the total variance of the time series (Refinetti, 2004). Robustness .14% is higher than the noise level and indicates statistically significant rhythmicity. One-way ANOVA was applied to determine statistically significant differences in the rhythmic parameters among
42
Journal of Veterinary Behavior, Vol 8, No 1, January/February 2013
Figure 1 Summary of experimental conditions. White and black bars indicate photophase and scotophase. *Food administration. YDay of total locomotor activity recording.
the feeding schedules. All housing and care conformed to the standards recommended by the Guide for the Care and Use of Laboratory Animals and Directive 86/609 CEE.
Results Visual inspection of actograms showed that total locomotor activity was not evenly distributed throughout the day, but was mainly diurnal, except during the fasting period. Figure 2 shows a representative actogram of the total locomotor activity recorded in a pony during the 5 experimental periods. Statistical significant difference in the amount of activity between photophase and scotophase (P , 0.0001; F(4,50) 5 31.20) was observed in all experimental periods.
Higher levels of activity were observed during the photophase (Figure 2). There was no difference in the amount of activity recorded within each photophase across all feeding schedules. The same condition was observed for the amount of activity recorded within each scotophase. Twoway ANOVA revealed a significant (F(24,600) 5 2.46; P , 0.0001) main effect of the time of day and no effect of feeding schedules on the amount of total locomotor activity. Ponies spent about 13 h/d to completely consume 4 kg of hay when provided once a day, and about 6 hours to completely consume of 2 kg of hay when provided twice. The application of the periodic model and statistical analysis of the cosinor enabled us to define the periodic parameters. Mesor values and acrophase did not change among the experimental periods. Acrophase was always
Figure 2 Actograms of total locomotor activity record in a pony subjected to a 12:12-h light/dark cycle and different feeding schedules. White and black bars indicate photophase and scotophase.
Piccione et al
Daily rhythm of total locomotor activity in ponies
Figure 3 Mean 6 standard error of mean (SEM) of acrophases observed during the different experimental periods. White and black bars indicate photophase and scotophase.
diurnal (Figure 3). The amplitude of the rhythm statistically changed among the experimental periods (P , 0.002) and was statistically lower (P , 0.05) during the fasting period than the other ones (Figure 4). Robustness was influenced by experimental periods (P , 0.0002): it was higher when fed once a day at 8 PM and when fed once a day at 8 AM than the other ones (Figure 4).
Discussion Our results showed that the amount of daily total locomotor activity in ponies is not influenced by the time of food administration. In accordance with a previous study in which locomotor activity peaked in the middle of the light phase in trained and untrained horses (Piccione et al., 2011), higher levels of activity were observed in all experimental periods during the photophase in our study, and there were no differences between the amount of activity recorded within each photophase and scotophase across all feeding schedules. Although animals always received the same amount of ration (4 kg/d) that meets the absolute amount of nutrition daily and simulates an ad libitum administration, the different feeding schedules induced a different distribution of activity between all feeding schedules. In particular, during the fasting period, it was spread through the day compared with the other periods (Figure 2). In most cases, feeding schedules appear to entrain only a component of activity, which anticipates the daily feeding time. When food availability is limited to a several-hour interval at a particular time each day, mammals quickly develop a new component of daily behavioral activityda second period of arousal and increased locomotor activity that occurs shortly before the time of daily food presentation (Mistlberger, 1994). This component, known as food anticipatory activity (FAA), exhibits fundamental properties of a clock-controlled process, including resistance to entrainment and persistence in the absence of the Zeitgeber (Mistlberger, 1994). It is thought that FAA
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represents an evolutionary adaptation providing mammals with a highly flexible and efficient food-seeking program, one that is able, if necessary, to take advantage of food sources available at unusual times with respect to the animal’s typical daily rest–activity cycle (Storch and Weitz, 2009). In our study, FAA failed to emerge in all days tested. Replenishing food hoppers at the same time each day without caloric restriction does not induce food anticipation (Mistlberger, 2009). Ponies self-regulated their intake over the 24 hours. The physical form in which a feed is presented alters the rate of consumption and total dry matter intakes by horses and ponies. Both foraging and eating behavior are modified to allow optimal rates of food ingestion (Arnold, 1985). Horses distribute their foraging and feeding behavior across day and night (Martin et al., 2010). Their digestive system is designed for a small intake at frequent intervals. In the study by Ralston (1984), the horses were given free access to feed, and although confined to stalls, they engaged in meal patterns that were similar to those described under free ranging conditions. Stable conditions can influence the locomotor activity intensity, but not the feeding behavior (Piccione et al., 2008). Food has been considered a dominant Zeitgeber for peripheral clocks in various mammalian species (Damiola et al., 2000). Changes in the circadian pattern of eating and time spent eating are the behavioral response of the animal to the amount of food availability. The daily routine of maintenance behavior is determined by the timing of feeding, with other activities accommodated to it (Arnold, 1985). The overall rhythmic pattern of mares maintained outdoors in a large 4-acre pasture was both strongly ultradian and weakly circadian (Martin et al., 2010). In particular, the different time of food administration influenced the amplitude and the robustness of the rhythm. The absence of feeding behavior during the fasting period determined a statistical decrease of the amplitude of the daily rhythm of locomotor activity. The amplitude of the rhythms can be affected by many factors downstream from the oscillator responsible for timing the rhythms. If phase, precision, and period are unaltered, a change in amplitude is probably an effect downstream from the clock (Mistlberger, 2009). A more robust rhythm was observed during the period when the animals were fed once a day, and providing hay at 8 PM did not invert the feeding behavior. In the period when the animals were fed once at 8 PM, we might have expected an increase of activity during the scotophase after providing the food, whereas the animals maintained their feeding behavior and a diurnal activity. In mammals, it appears that the unresponsiveness of the SCN clock to feeding time reflects an inherent property of the central pacemaker, rather than a dominance of photic entrainment over food entrainment (Damiola et al., 2000). In the presence of a daily L/D cycle, the phase of the SCN is altered relatively little or not at all by daily feeding schedules, regardless of mealtime (Damiola et al., 2000; Stokkan et al., 2001).
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Journal of Veterinary Behavior, Vol 8, No 1, January/February 2013
Figure 4 Mean 6 standard error of mean (SEM) of the robustness and amplitude of the rhythm observed during the different experimental periods. *P , 0.05 versus other periods.
Ponies can distribute their activity in different components, without significantly modifying daily total activity according to the response to external stimuli, such as food availability.
Conclusion Our findings support the theory that when given essentially free choice hay, the time of feeding did not entrain activity, underlining a dominance of photic entrainment over food entrainment. Therefore, feeding behavior can influence the distribution of locomotor activity during a 24-hour period in ponies, with changes in the amplitude and robustness of the rhythm. These findings underline that care must always be taken in considering that feeding schedules must be adequate to ensure the physiological rhythm of the animals.
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