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Daily rhythms of rectal temperature and total locomotor activity in trained and untrained horses
Journal of Veterinary Behavior (2011) 6, 115-120
RESEARCH
Daily rhythms of rectal temperature and total locomotor activity in trained and untrained horses Giuseppe Piccione, Claudia Giannetto, Simona Marafioti, Stefania Casella, Francesco Fazio, Giovanni Caola Department of Experimental Science and Applied Biotechnology, Laboratory of Veterinary Chronophysiology, Faculty of Veterinary Medicine, University of Messina, Messina, Italy. KEYWORDS: body temperature; locomotor activity; sedentary; athletes; horse
Abstract In this study the authors evaluated the influence of physical activity on the daily rhythm of rectal temperature and total locomotor activity in untrained and trained horses. Rectal temperature and locomotor activity of 12 Italian saddle horses, 6 untrained (group A) and 6 trained (group B), was recorded for 48 h. Rectal temperature was recorded every 4 h with a rectal probe. Animals were equipped with actigraphy-based data loggers, Actiwatch-MiniÒ to record total activity. The application of twoway ANOVA showed a highly significant effect of time and exercise on rectal temperature in untrained and trained horses in both days of monitoring. A significant effect of time on locomotor activity was observed, but there was no effect of exercise. Cosinor analysis identified the periodic parameters and their acrophases during the 2 days of monitoring. For all rhythmic parameters of rectal temperature except amplitude, no statistically significant differences were observed between the two groups. Statistically significant differences for the rhythmic parameters of locomotor activity, except robustness, between untrained and trained horses were found. In conclusion, rectal temperature circadian pattern was similar in untrained and trained horses, indicating that the endogenous nature of its rhythm was not influenced by external stimuli such as physical exercise. Ó 2011 Elsevier Inc. All rights reserved.
Introduction Circadian rhythms are an inherent property of living systems and constitute an essential part of their internal and external temporal order. Rhythm generation is realized by a complex system with the central pacemaker within the suprachiasmatic nuclei (Weinert and Waterhouse, 2007; Mongrain and Cermakian, 2009). Address reprint requests and correspondence: Giuseppe Piccione, Department of Experimental Science and Applied Biotechnology, Laboratory of Veterinary Chronophysiology, Faculty of Veterinary Medicine, University of Messina, 98168 Messina, Italy; Tel: 139 0903503584; Fax: 139 0903503975. E-mail: [email protected] 1558-7878/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jveb.2010.11.003
The existence of daily or circadian rhythms across variables in number of species has been documented (Piccione et al., 2003). In horses, different physiological variables show different degrees of daily rhythmicity and reach their daily peaks at different times of the day (Piccione et al., 2005). Previous study conducted in horses and sheep showed that most variables, such as locomotor activity, peaked during the light phase of the light–dark, whereas others, such as rectal temperature, peaked during scotophase (Piccione et al., 2005). Rectal temperature is widely studied and its ultradian and circadian rhythms are exhibited in many mammals (Refinetti and Menaker, 1992). Rectal temperature is regulated within narrow limits by a complex feedback system.
116 Body temperature and its circadian rhythm are important variables for many reasons. The homeostatic regulation of body temperature is a fundamental physiological process that ensures the stability of the internal milieu in mammals. Regulation of body temperature is an essential component in the process of fever and in the response of an organism to infection and disease (Piccione and Refinetti, 2003). Because of the relative ease of monitoring body temperature and the robustness of its rhythm, the rhythmicity of body temperature has been widely used as an indicator of the rhythmicity of the biological clock (Zulley et al., 1981; Klerman et al., 2002) and as an indicator of the general health of an animal and of its energy metabolism (Cossins and Bowler, 1997; Blumberg, 2002). Similarly, it is believed that locomotor activity ensures an optimal functioning of the biological system, with maximum efficiency, performance, and welfare (Piccione et al., 2010). In domestic animals variations in temperature are usually attributed to environmental inputs, feeding, and activity patterns (Hahn, 1989). Moreover, the rhythm in core temperature is promoted by behavioral changes (Weinert and Waterhouse, 2007). In diurnal species, these include increases in activity during the morning and decreases during the evening; also there is a preference for warmer ambient temperature in the morning and cooler ones in the evening (Refinetti, 1998). The thermoregulatory system is an encompassing system that uses behavioral and autonomic process in integration with other physiological systems, such as the respiratory system, the cardiovascular system, and the motor system (Piccione and Refinetti, 2003). Daily activity patterns have been well described in domestic mammals including rabbits, cats, dogs, sheep (Refinetti, 2006; Piccione et al., 2010), goats (Piccione et al., 2008a; Piccione et al., 2008b; Piccione et al., 2008c), and horses (Gill, 1991; Scheibe et al., 1999; Piccione et al., 2008d). Total locomotor activity that includes different behaviors, such as feeding, drinking, walking, grooming, and small movement during sleep, is influenced by different factors such as photoperiod (Bertolucci et al., 2008), different stabling conditions (Piccione et al., 2008e), and feeding schedules (Piccione et al., 2007). It is now clear that locomotor activity in mammals is controlled by both endogenous circadian rhythms and acute light exposure (Aschoff, 1960; Berger, 2008). Light acutely suppresses locomotor activity in nocturnal mammals, but promotes activity in diurnal mammals. The direct effect of light on activity is mediated by a nonimage-forming visual pathway, starting with melanopsin expressing intrinsically photoreceptive retinal ganglion cells (Hattar et al., 2003; Panda et al., 2003). This phenomenon, known as masking, can countermand circadian signals regulating activity levels and constitutes a parallel system for matching behavioral states with the diurnal cycle. Thus, the rhythm of core temperature is correlated with rhythms of sleep (Dijk and Czeisler, 1995), physical performance, and mental performance (Waterhouse et al., 2001a, 2005). The sleep–wake cycle may have a direct effect on
Journal of Veterinary Behavior, Vol 6, No 2, March/April 2011 the temperature rhythm, with sleep and its associated change in posture resulting in decrease in temperature, and both physical and mental activities resulting in increase in temperature (Waterhouse et al., 1995, 2001b). An individual normally falls asleep when core temperature is decreasing, and the main sleep period ends on the rising part of the circadian temperature curve. Extending the study of rhythmicity of body temperature to farm animals is important not only from a comparative perspective (as most studies conducted so far concentrated on human beings and laboratory animals), but also from an economic perspective (as greater knowledge of this process can lead to improvements in livestock production practices). Because the correlation between body temperature and locomotor activity has been well studied only in human beings and small mammals, the aim of this study was to investigate these 2 variables simultaneously and to evaluate the influence of physical activity on the daily rhythm of total locomotor activity and body temperature in untrained and trained horses.
Materials and Methods Animals A total of 12 Sella Italian saddle horses (females, 8-10 years old, 530 6 20 kg body weight) were divided into 2 groups of 6 untrained (group A) and 6 regularly trained (group B) animals. All horses were subjected to the same type of management and were housed in individual boxes under natural spring photoperiod (sunrise at 06:00 hours, sunset at 19:00 hours), at an indoor temperature of 18-21 C, with feeding (hay and oat) provided 3 times a day, at 07:00, at 12:00, and 18:00 hours, and water available ad libitum. Before the start of the study, the untrained horses were always kept inside their box (4 m ! 4 m) equipped with a paddock (20 m ! 20 m) for 60 days, and none of them were subjected to physical exercise; in contrast, the group of trained horses underwent fitness training 6 days a week, with a rest day on Sundays. Training started at 15:30 every day, lasting for 1 hour. Four days a week, the 1 hour of training included 5 minutes of walking, 25 minutes of trotting, 25 minutes of galloping, and 5 minutes of walking; during the maximal physical effort, the heart rate did not exceed the 120 beats/min. The 1 hour of training included 5 minutes of walking, 20 minutes of trotting, 15 minutes of galloping, jumping exercise, and 5 minutes of walking for 2 days a week; during the maximal physical effort, the heart rate did not exceed the 180 beats/min. Fitness training and animal general care were performed by a professional staff not associated with the research team.
Data collection Rectal temperature was recorded every 4 hours for 48 hours, starting at 07:00 hours on day 1 and finishing at
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Daily rhythms of rectal temperature and total locomotor activity
07:00 hours on day 3 in both groups. The digital thermometer probe (model HI92704, Hanna Instruments, Rome, Italy) had a resolution of 0.1 C, was inserted 15 cm into the rectum. Total locomotor activity of horses, which included feeding, drinking, walking, grooming, and small movements during sleep, was recorded for 2 days of experimental period. Actiwatch-MiniÒ (Cambridge Neurotechnology Ltd, Cambridge, UK), actigraphy-based data loggers that records a digitally integrated measure of motor activity, was placed on each horse. This activity acquisition system is based on miniaturized accelerometer technologies, currently used for human activity monitoring, but also tested for activity monitoring in small nonhuman mammals (Munoz-Delgrado et al., 2004; Mann et al., 2005). Actiwatch uses a piezo-electric accelerometer that is set up to record the integration of intensity, amount, and duration of movement in all directions. The corresponding voltage produced is converted and stored as an activity count in the memory unit of the Actiwatch. The maximum sampling frequency is 32 Hz. Actigraphs were placed by means of collars that were accepted without any apparent disturbance. Activity was monitored with a sampling interval of 5 minutes. Actograms, a type of graph commonly used in circadian research to plot activity against time, were drawn using Actiwatch Activity Analysis 5.06 (Cambridge Neurotechnology Ltd, UK). Total daily amount of activity and the amount of activity during the photophase and the scotophase were calculated using Actiwatch Activity Analysis 5.06. The Cosine peak of a rhythm (i.e., the time of the daily peak) was competed by cosinor rhythmometry (Nelson, 1979) as implement in the Actiwatch Activity Analysis 5.06 program.
Statistical analysis Two-way analysis of variance was used to determine statistically significant modifications caused by the difference in experimental conditions (at the significance level 2 alpha 5 0.05). Four rhythmic parameters were determined for each of the 2 variables in each horse: mean level, amplitude, acrophase (time of peak), and robustness (strength of rhythmicity). The amplitude of a rhythm was calculated as half the range of oscillation, which on its turn was computed as the difference between peak and trough. The acrophase of a rhythm was determined by an iterative curve-fitting procedure based on the single cosinor procedure, as described by Nelson et al. (1979). Rhythm robustness was computed as a percentage of the maximal score attained by the chi-square periodogram statistic for ideal datasets of comparable size and 24-hour periodicity (Refinetti, 2004). Robustness greater than 20% is above noise level and indicates statistically significant rhythmicity. Unpaired Student’s t-test was used to evaluate statistical differences between rhythmic parameters of the 2 variables
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Figure 1 Patterns of rectal temperature (line) and locomotor activity (dotted area) in untrained (group A) and trained (group B) horses. White and black bars indicate photophase and scotophase.
studied in untrained and trained horses. Regression lines between mean values of rectal temperature and locomotor activity with 95% confidence intervals and the correlation coefficient (r) were evaluated. All the work presented in this study complies with current international guidelines on the use and care of animals.
Results The results obtained during the experimental period indicated the existence of a daily rhythm of rectal temperature and locomotor activity in both groups, during 48 hours of monitoring. The rhythm of rectal temperature peaked early in the dark phase; the locomotor activity peaked in the middle of the light phase (Figure 1). The application of 2way analysis of variance showed a highly significant effect of time and exercise on rectal temperature in untrained and trained horses in both days of monitoring (Time: day 1 F(6,60) 5 42.82, P , 0.0001; day 2 F(6,60) 5 58.94, P , 0.0001; Physical exercise: day 1 F(1,60) 5 8.848, P , 0.05, day 2 F(1,60) 5 5.302, P , 0.05,). A significant effect of time on locomotor activity was observed, but no
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Discussion Rhythmic parameters of rectal temperature found in this study are in agreement with those observed previously. It was observed that the temperature rhythm both in untrained than in trained horses peaked early in the dark phase with acrophases between 18:56 and 19:30 (Piccione et al., 2008d). Furthermore, our results are in accordance to other studies in which horses showed a stable diurnal daily rhythm of locomotor activity characterized by a high robustness and high diurnality index. Although 2 groups were kept under different housing conditions that altered the daily activity patterns (Piccione et al., 2008e), the application of exercise in the present study abolished this alteration. The simultaneous study of many variables is necessary to understand multiple temporal relationships of physiological processes. In most studies of small animals, where body temperature and locomotor activity were measured simultaneously, the 2 variables had very similar circadian rhythms with the high phase of the body temperature occurring during the active phase of the circadian rhythm of locomotor activity (Refinetti and Menaker, 1992). In contrast, our study showed that in horses, rectal temperature and locomotor activity have different circadian rhythms and increased locomotor activity does not lead to an increase in rectal temperature. The fact that acute episodes of physical activity and exercise can elevate body temperature has been extensively documented in studies conducted in both human beings (Fujishima, 1986; Gander et al., 1986) and animals (Paladino et al., 1984; Tanaka et al., 1990). Consequently, daily variation in the level of activity could, in principle, be the cause of circadian rhythm of
400
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effect of exercise was observed (Time: day 1 F(24,240) 5 51.05, P , 0.0001; day 2 F(24,240) 5 11,64, P , 0.0001). The application of the periodic model and statistical analysis of the cosinor enabled us to define the periodic parameters and their acrophases during the 2 days of monitoring (Figure 2). In both groups rectal temperature showed nocturnal acrophases. The acrophases were observed at 21:50 and 22:25 in group A, and 20:50 and 21:58 in group B during the days 1 and 2, respectively. In contrast, locomotor activity showed diurnal acrophases, observed at 16:05 and 16:39 in group A, and 13:40 and 13:58 in group B during the days 1 and 2, respectively. Unpaired Student’s t-test did not show statistically significant differences between untrained and trained horses for any rhythmic parameters of rectal temperature except amplitude; amplitude was higher in trained than untrained horses (P , 0.04). The rhythmic parameters of locomotor activity showed statistically significant differences between untrained and trained horses’, robustness was higher in untrained than trained horses. No significant linear correlation among temperature values and locomotor activity was found.
Journal of Veterinary Behavior, Vol 6, No 2, March/April 2011
Rectal temperature ( °C)
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Figure 2 Mean values (6standard deviation) of 4 rhythmic parameters of rectal temperature and total locomotor activity recorded during 48 hours of monitoring.
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Daily rhythms of rectal temperature and total locomotor activity
body temperature. In a previous study performed in small mammals, it was found that the rhythm of body temperature was consistently more robust than the rhythm of locomotor activity (Refinetti, 1999). Conceptually, a rhythm with a low robustness cannot be the cause of a rhythm with high robustness. Many observations indicated that the circadian rhythm of body temperature is not a consequence of the circadian rhythm of activity. Studies of human subjects who are in constant bed rest (Marotte and Timbal, 1981; Krauchi and Wirz-Justice, 1994; Monk et al., 1996; Carrier and Monk, 1997; Murray et al., 2002) and correlation studies in animals (Bolles et al., 1968; Honma and Hiroshige, 1978; Refinetti, 1994; Gordon and Yang, 1997; Refinetti, 1999) have clearly shown that the temperature rhythm is autonomous, although affected by the activity rhythm. In addition to studies in which the circadian rhythm of activity was experimentally blocked to demonstrate the autonomy of the circadian rhythm of body temperature, studies under natural conditions also provided evidence for the independence of the 2 rhythms. Morning types (i.e., people who usually wake up early and perform, optimally early in the day) and evening types were found to have identical parameters of the circadian rhythm of body temperature despite the difference in the phase of their activity rhythm (Lattanzi et al., 1988). We showed that rectal temperature rhythm reaches its acrophase in scotophase, whereas the locomotor activity rhythm reaches its acrophase in the middle of photophase. This relationship between the 2 rhythms resembles that found in human beings, other breeds of horses, and sheep, who are active during most of the day but whose temperature rhythm reaches the daily peak in the photophase (Murray et al., 2002; Piccione et al., 2005). The difference in the robustness of rhythms and acrophases of the parameters studied is probably because peripheral tissue clocks can manifest a remarkable independence from so-called master clocks, particularly in animals with lightentrainable peripheral oscillators (Schibler and SassoneCorsi, 2002). Previous study showed that rectal temperature rhythm persisted in horses maintained under constant illumination and thus confirmed the endogenous nature of this rhythm (Piccione et al., 2002). In conclusion, we can claim that, in horses, there is no correlation between rectal temperature and activity level and we can confirm that rectal temperature maintained a similar circadian pattern regardless of exercise, indicating that the endogenous nature of its rhythm was not influenced by external stimuli such as physical exercise.
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RESEARCH
Daily rhythms of rectal temperature and total locomotor activity in trained and untrained horses Giuseppe Piccione, Claudia Giannetto, Simona Marafioti, Stefania Casella, Francesco Fazio, Giovanni Caola Department of Experimental Science and Applied Biotechnology, Laboratory of Veterinary Chronophysiology, Faculty of Veterinary Medicine, University of Messina, Messina, Italy. KEYWORDS: body temperature; locomotor activity; sedentary; athletes; horse
Abstract In this study the authors evaluated the influence of physical activity on the daily rhythm of rectal temperature and total locomotor activity in untrained and trained horses. Rectal temperature and locomotor activity of 12 Italian saddle horses, 6 untrained (group A) and 6 trained (group B), was recorded for 48 h. Rectal temperature was recorded every 4 h with a rectal probe. Animals were equipped with actigraphy-based data loggers, Actiwatch-MiniÒ to record total activity. The application of twoway ANOVA showed a highly significant effect of time and exercise on rectal temperature in untrained and trained horses in both days of monitoring. A significant effect of time on locomotor activity was observed, but there was no effect of exercise. Cosinor analysis identified the periodic parameters and their acrophases during the 2 days of monitoring. For all rhythmic parameters of rectal temperature except amplitude, no statistically significant differences were observed between the two groups. Statistically significant differences for the rhythmic parameters of locomotor activity, except robustness, between untrained and trained horses were found. In conclusion, rectal temperature circadian pattern was similar in untrained and trained horses, indicating that the endogenous nature of its rhythm was not influenced by external stimuli such as physical exercise. Ó 2011 Elsevier Inc. All rights reserved.
Introduction Circadian rhythms are an inherent property of living systems and constitute an essential part of their internal and external temporal order. Rhythm generation is realized by a complex system with the central pacemaker within the suprachiasmatic nuclei (Weinert and Waterhouse, 2007; Mongrain and Cermakian, 2009). Address reprint requests and correspondence: Giuseppe Piccione, Department of Experimental Science and Applied Biotechnology, Laboratory of Veterinary Chronophysiology, Faculty of Veterinary Medicine, University of Messina, 98168 Messina, Italy; Tel: 139 0903503584; Fax: 139 0903503975. E-mail: [email protected] 1558-7878/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jveb.2010.11.003
The existence of daily or circadian rhythms across variables in number of species has been documented (Piccione et al., 2003). In horses, different physiological variables show different degrees of daily rhythmicity and reach their daily peaks at different times of the day (Piccione et al., 2005). Previous study conducted in horses and sheep showed that most variables, such as locomotor activity, peaked during the light phase of the light–dark, whereas others, such as rectal temperature, peaked during scotophase (Piccione et al., 2005). Rectal temperature is widely studied and its ultradian and circadian rhythms are exhibited in many mammals (Refinetti and Menaker, 1992). Rectal temperature is regulated within narrow limits by a complex feedback system.
116 Body temperature and its circadian rhythm are important variables for many reasons. The homeostatic regulation of body temperature is a fundamental physiological process that ensures the stability of the internal milieu in mammals. Regulation of body temperature is an essential component in the process of fever and in the response of an organism to infection and disease (Piccione and Refinetti, 2003). Because of the relative ease of monitoring body temperature and the robustness of its rhythm, the rhythmicity of body temperature has been widely used as an indicator of the rhythmicity of the biological clock (Zulley et al., 1981; Klerman et al., 2002) and as an indicator of the general health of an animal and of its energy metabolism (Cossins and Bowler, 1997; Blumberg, 2002). Similarly, it is believed that locomotor activity ensures an optimal functioning of the biological system, with maximum efficiency, performance, and welfare (Piccione et al., 2010). In domestic animals variations in temperature are usually attributed to environmental inputs, feeding, and activity patterns (Hahn, 1989). Moreover, the rhythm in core temperature is promoted by behavioral changes (Weinert and Waterhouse, 2007). In diurnal species, these include increases in activity during the morning and decreases during the evening; also there is a preference for warmer ambient temperature in the morning and cooler ones in the evening (Refinetti, 1998). The thermoregulatory system is an encompassing system that uses behavioral and autonomic process in integration with other physiological systems, such as the respiratory system, the cardiovascular system, and the motor system (Piccione and Refinetti, 2003). Daily activity patterns have been well described in domestic mammals including rabbits, cats, dogs, sheep (Refinetti, 2006; Piccione et al., 2010), goats (Piccione et al., 2008a; Piccione et al., 2008b; Piccione et al., 2008c), and horses (Gill, 1991; Scheibe et al., 1999; Piccione et al., 2008d). Total locomotor activity that includes different behaviors, such as feeding, drinking, walking, grooming, and small movement during sleep, is influenced by different factors such as photoperiod (Bertolucci et al., 2008), different stabling conditions (Piccione et al., 2008e), and feeding schedules (Piccione et al., 2007). It is now clear that locomotor activity in mammals is controlled by both endogenous circadian rhythms and acute light exposure (Aschoff, 1960; Berger, 2008). Light acutely suppresses locomotor activity in nocturnal mammals, but promotes activity in diurnal mammals. The direct effect of light on activity is mediated by a nonimage-forming visual pathway, starting with melanopsin expressing intrinsically photoreceptive retinal ganglion cells (Hattar et al., 2003; Panda et al., 2003). This phenomenon, known as masking, can countermand circadian signals regulating activity levels and constitutes a parallel system for matching behavioral states with the diurnal cycle. Thus, the rhythm of core temperature is correlated with rhythms of sleep (Dijk and Czeisler, 1995), physical performance, and mental performance (Waterhouse et al., 2001a, 2005). The sleep–wake cycle may have a direct effect on
Journal of Veterinary Behavior, Vol 6, No 2, March/April 2011 the temperature rhythm, with sleep and its associated change in posture resulting in decrease in temperature, and both physical and mental activities resulting in increase in temperature (Waterhouse et al., 1995, 2001b). An individual normally falls asleep when core temperature is decreasing, and the main sleep period ends on the rising part of the circadian temperature curve. Extending the study of rhythmicity of body temperature to farm animals is important not only from a comparative perspective (as most studies conducted so far concentrated on human beings and laboratory animals), but also from an economic perspective (as greater knowledge of this process can lead to improvements in livestock production practices). Because the correlation between body temperature and locomotor activity has been well studied only in human beings and small mammals, the aim of this study was to investigate these 2 variables simultaneously and to evaluate the influence of physical activity on the daily rhythm of total locomotor activity and body temperature in untrained and trained horses.
Materials and Methods Animals A total of 12 Sella Italian saddle horses (females, 8-10 years old, 530 6 20 kg body weight) were divided into 2 groups of 6 untrained (group A) and 6 regularly trained (group B) animals. All horses were subjected to the same type of management and were housed in individual boxes under natural spring photoperiod (sunrise at 06:00 hours, sunset at 19:00 hours), at an indoor temperature of 18-21 C, with feeding (hay and oat) provided 3 times a day, at 07:00, at 12:00, and 18:00 hours, and water available ad libitum. Before the start of the study, the untrained horses were always kept inside their box (4 m ! 4 m) equipped with a paddock (20 m ! 20 m) for 60 days, and none of them were subjected to physical exercise; in contrast, the group of trained horses underwent fitness training 6 days a week, with a rest day on Sundays. Training started at 15:30 every day, lasting for 1 hour. Four days a week, the 1 hour of training included 5 minutes of walking, 25 minutes of trotting, 25 minutes of galloping, and 5 minutes of walking; during the maximal physical effort, the heart rate did not exceed the 120 beats/min. The 1 hour of training included 5 minutes of walking, 20 minutes of trotting, 15 minutes of galloping, jumping exercise, and 5 minutes of walking for 2 days a week; during the maximal physical effort, the heart rate did not exceed the 180 beats/min. Fitness training and animal general care were performed by a professional staff not associated with the research team.
Data collection Rectal temperature was recorded every 4 hours for 48 hours, starting at 07:00 hours on day 1 and finishing at
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07:00 hours on day 3 in both groups. The digital thermometer probe (model HI92704, Hanna Instruments, Rome, Italy) had a resolution of 0.1 C, was inserted 15 cm into the rectum. Total locomotor activity of horses, which included feeding, drinking, walking, grooming, and small movements during sleep, was recorded for 2 days of experimental period. Actiwatch-MiniÒ (Cambridge Neurotechnology Ltd, Cambridge, UK), actigraphy-based data loggers that records a digitally integrated measure of motor activity, was placed on each horse. This activity acquisition system is based on miniaturized accelerometer technologies, currently used for human activity monitoring, but also tested for activity monitoring in small nonhuman mammals (Munoz-Delgrado et al., 2004; Mann et al., 2005). Actiwatch uses a piezo-electric accelerometer that is set up to record the integration of intensity, amount, and duration of movement in all directions. The corresponding voltage produced is converted and stored as an activity count in the memory unit of the Actiwatch. The maximum sampling frequency is 32 Hz. Actigraphs were placed by means of collars that were accepted without any apparent disturbance. Activity was monitored with a sampling interval of 5 minutes. Actograms, a type of graph commonly used in circadian research to plot activity against time, were drawn using Actiwatch Activity Analysis 5.06 (Cambridge Neurotechnology Ltd, UK). Total daily amount of activity and the amount of activity during the photophase and the scotophase were calculated using Actiwatch Activity Analysis 5.06. The Cosine peak of a rhythm (i.e., the time of the daily peak) was competed by cosinor rhythmometry (Nelson, 1979) as implement in the Actiwatch Activity Analysis 5.06 program.
Statistical analysis Two-way analysis of variance was used to determine statistically significant modifications caused by the difference in experimental conditions (at the significance level 2 alpha 5 0.05). Four rhythmic parameters were determined for each of the 2 variables in each horse: mean level, amplitude, acrophase (time of peak), and robustness (strength of rhythmicity). The amplitude of a rhythm was calculated as half the range of oscillation, which on its turn was computed as the difference between peak and trough. The acrophase of a rhythm was determined by an iterative curve-fitting procedure based on the single cosinor procedure, as described by Nelson et al. (1979). Rhythm robustness was computed as a percentage of the maximal score attained by the chi-square periodogram statistic for ideal datasets of comparable size and 24-hour periodicity (Refinetti, 2004). Robustness greater than 20% is above noise level and indicates statistically significant rhythmicity. Unpaired Student’s t-test was used to evaluate statistical differences between rhythmic parameters of the 2 variables
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Figure 1 Patterns of rectal temperature (line) and locomotor activity (dotted area) in untrained (group A) and trained (group B) horses. White and black bars indicate photophase and scotophase.
studied in untrained and trained horses. Regression lines between mean values of rectal temperature and locomotor activity with 95% confidence intervals and the correlation coefficient (r) were evaluated. All the work presented in this study complies with current international guidelines on the use and care of animals.
Results The results obtained during the experimental period indicated the existence of a daily rhythm of rectal temperature and locomotor activity in both groups, during 48 hours of monitoring. The rhythm of rectal temperature peaked early in the dark phase; the locomotor activity peaked in the middle of the light phase (Figure 1). The application of 2way analysis of variance showed a highly significant effect of time and exercise on rectal temperature in untrained and trained horses in both days of monitoring (Time: day 1 F(6,60) 5 42.82, P , 0.0001; day 2 F(6,60) 5 58.94, P , 0.0001; Physical exercise: day 1 F(1,60) 5 8.848, P , 0.05, day 2 F(1,60) 5 5.302, P , 0.05,). A significant effect of time on locomotor activity was observed, but no
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Discussion Rhythmic parameters of rectal temperature found in this study are in agreement with those observed previously. It was observed that the temperature rhythm both in untrained than in trained horses peaked early in the dark phase with acrophases between 18:56 and 19:30 (Piccione et al., 2008d). Furthermore, our results are in accordance to other studies in which horses showed a stable diurnal daily rhythm of locomotor activity characterized by a high robustness and high diurnality index. Although 2 groups were kept under different housing conditions that altered the daily activity patterns (Piccione et al., 2008e), the application of exercise in the present study abolished this alteration. The simultaneous study of many variables is necessary to understand multiple temporal relationships of physiological processes. In most studies of small animals, where body temperature and locomotor activity were measured simultaneously, the 2 variables had very similar circadian rhythms with the high phase of the body temperature occurring during the active phase of the circadian rhythm of locomotor activity (Refinetti and Menaker, 1992). In contrast, our study showed that in horses, rectal temperature and locomotor activity have different circadian rhythms and increased locomotor activity does not lead to an increase in rectal temperature. The fact that acute episodes of physical activity and exercise can elevate body temperature has been extensively documented in studies conducted in both human beings (Fujishima, 1986; Gander et al., 1986) and animals (Paladino et al., 1984; Tanaka et al., 1990). Consequently, daily variation in the level of activity could, in principle, be the cause of circadian rhythm of
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effect of exercise was observed (Time: day 1 F(24,240) 5 51.05, P , 0.0001; day 2 F(24,240) 5 11,64, P , 0.0001). The application of the periodic model and statistical analysis of the cosinor enabled us to define the periodic parameters and their acrophases during the 2 days of monitoring (Figure 2). In both groups rectal temperature showed nocturnal acrophases. The acrophases were observed at 21:50 and 22:25 in group A, and 20:50 and 21:58 in group B during the days 1 and 2, respectively. In contrast, locomotor activity showed diurnal acrophases, observed at 16:05 and 16:39 in group A, and 13:40 and 13:58 in group B during the days 1 and 2, respectively. Unpaired Student’s t-test did not show statistically significant differences between untrained and trained horses for any rhythmic parameters of rectal temperature except amplitude; amplitude was higher in trained than untrained horses (P , 0.04). The rhythmic parameters of locomotor activity showed statistically significant differences between untrained and trained horses’, robustness was higher in untrained than trained horses. No significant linear correlation among temperature values and locomotor activity was found.
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Figure 2 Mean values (6standard deviation) of 4 rhythmic parameters of rectal temperature and total locomotor activity recorded during 48 hours of monitoring.
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body temperature. In a previous study performed in small mammals, it was found that the rhythm of body temperature was consistently more robust than the rhythm of locomotor activity (Refinetti, 1999). Conceptually, a rhythm with a low robustness cannot be the cause of a rhythm with high robustness. Many observations indicated that the circadian rhythm of body temperature is not a consequence of the circadian rhythm of activity. Studies of human subjects who are in constant bed rest (Marotte and Timbal, 1981; Krauchi and Wirz-Justice, 1994; Monk et al., 1996; Carrier and Monk, 1997; Murray et al., 2002) and correlation studies in animals (Bolles et al., 1968; Honma and Hiroshige, 1978; Refinetti, 1994; Gordon and Yang, 1997; Refinetti, 1999) have clearly shown that the temperature rhythm is autonomous, although affected by the activity rhythm. In addition to studies in which the circadian rhythm of activity was experimentally blocked to demonstrate the autonomy of the circadian rhythm of body temperature, studies under natural conditions also provided evidence for the independence of the 2 rhythms. Morning types (i.e., people who usually wake up early and perform, optimally early in the day) and evening types were found to have identical parameters of the circadian rhythm of body temperature despite the difference in the phase of their activity rhythm (Lattanzi et al., 1988). We showed that rectal temperature rhythm reaches its acrophase in scotophase, whereas the locomotor activity rhythm reaches its acrophase in the middle of photophase. This relationship between the 2 rhythms resembles that found in human beings, other breeds of horses, and sheep, who are active during most of the day but whose temperature rhythm reaches the daily peak in the photophase (Murray et al., 2002; Piccione et al., 2005). The difference in the robustness of rhythms and acrophases of the parameters studied is probably because peripheral tissue clocks can manifest a remarkable independence from so-called master clocks, particularly in animals with lightentrainable peripheral oscillators (Schibler and SassoneCorsi, 2002). Previous study showed that rectal temperature rhythm persisted in horses maintained under constant illumination and thus confirmed the endogenous nature of this rhythm (Piccione et al., 2002). In conclusion, we can claim that, in horses, there is no correlation between rectal temperature and activity level and we can confirm that rectal temperature maintained a similar circadian pattern regardless of exercise, indicating that the endogenous nature of its rhythm was not influenced by external stimuli such as physical exercise.
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