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State of the art on daily rhythms of physiology and behaviour in horses
Giuseppe Piccione* and Claudia Giannetto
Department of Experimental Sciences and Applied Biotechnologies, Laboratory of Veterinary
Chronophysiology, University of Messina, Messina, 98168, Italy
(Received 10 March 2010; final version received 2 May 2010)
This article reviews the literature on the daily rhythm in horses. Many
physiological processes have been studied in horses to investigate their daily
rhythmicity. In horses many rhythms are driven by an endogenous pacemaker,
some of them endogenously generated, others influenced by external stimuli. It
then addresses the influence of maturation and ageing, physical exercise and jet
lag on these rhythms. The study of daily rhythms in horses is of considerable
importance in all aspects of horse management to maintain the natural
physiological behaviours of this species, necessary to guarantee the good welfare
in breeding and athlete horses. Chronophysiology in horses also has clinical
implications, it can influence disease occurrence, diagnostic tests and the effect of
medical treatments.
Keywords: daily rhythm; horses; physiological parameters; behaviour
Introduction
Many biochemical, physiological and behavioural parameters cycle in 24–hour
intervals. Circadian rhythms reflect extensive programming of biological activity that
meets and exploits the challenges and opportunities offered by the periodic nature of
the environment. These programs offer clear advantages of anticipatory preparation
for predictably recurrent conditions (Pittendrigh 1993), and regulate the behavioural
and physiological rhythms that underlie adaptative partitioning of an organism’s
activities in ways that optimise survival.
The mammalian circadian timing system is composed of almost as many
individual clocks as there are cells. The countless oscillators have to be synchronised
by a central pacemaker to coordinate temporal physiology and behaviour (Schibler
and Sassone-Corsi 2002).
Synchronisation is of utmost importance for survival of the entire organism. To
ensure this, the central pacemaker located in the SCN of the hypothalamus imposes
temporal structure across the brain and peripheral organs via neural and endocrine
outputs (Holzberg and Albrecht 2003). The rhythms interact with each other as well
as the outside fluctuating, noisy environments under the control of innumerable
feedback systems that provide an orderly function that enables life (Glass 2001).
*Corresponding author. Email: giuseppe.piccione@unime.it
Biological Rhythm Research
Vol. 42, No. 1, February 2011, 67–88
ISSN 0929-1016 print/ISSN 1744-4179 online
� 2011 Taylor & Francis
DOI: 10.1080/09291016.2010.491247
http://www.informaworld.com
Synchronisation of peripheral clocks by the master clock in the SCN ensures that
each tissue can adapt its specific function to the correct time of day by means of
tissue-specific circadian regulation of transcription.
Circadian rhythmicity results from interactions between clock genes and clock
proteins. The levels of these proteins fluctuate in a rhythmic pattern, according to
translation of mRNA from clock genes, and it takes about 24–25 h for one such
cycle to be completed (Reilly and Waterhouse 2009). Proteins with timekeeping
functions have also been located in other organs, including heart, liver, kidneys,
endocrine glands and skeletal muscle, implying that peripheral as well as central
factors are engaged in the causal nexus of circadian rhythms (Oishi et al. 2005). The
molecular basis for these rhythms may rely on a negative feedback loop in which
clock proteins negatively regulate their own abundance or activity. This regulation
may occur both at the transcriptional and the post-transcriptional level (Brown and
Schibler 1999).
Observing animals integrated into their environment, it can be noted that
endogenous rhythms are usually not free-running. The time of the body clock is
masked, and the spontaneous biological rhythms are obliged by the exogenous cycles
to adjust their period in accordance. This means that the biological time innately has
the capacity to uniform itself with the physical time.
Various environmental factors act hierarchically as synchronisers of biological
rhythms. The most powerful synchroniser is the light–dark alternation. A rapid
change in time zones causes a desynchronisation between biological time and physical
time. The meal schedule is also a robust synchroniser. Subjects fed a complete meal
only once a day show a phase shift for many biological rhythms in relation to the time
of food administration. Social routines are also important, especially shift work. A
random shift can produce desynchronising effects for many periodic functions,
especially those related to physical performance. Other environmental agents causing
desynchronisation are stress, fasting, fatigue, etc., if abnormally prolonged in time
and/or cyclically repeated.
Different species have clearly distinct circadian periods, even though they live
in similar environments, genetic influences are stronger than environmental
influences. Many species can be classified as either diurnal (day-active) or
nocturnal (night-active). Therefore, the distinction between diurnality and
nocturnality is not always straightforward. Diurnal animals fill the space between
the peaks at dawn and dusk, while nocturnal animals fill the space between the
peaks at dusk and dawn. Crepuscular animals do not fill either space
preferentially so their rhythms are less robust (Kas and Edgar 1998; Refinetti
1999; Verhagen et al. 2004).
Daily rhythmicity is more robust in some organisms than in others, and more
robust in some physiological variables than in others. The magnitude and
consistency of the oscillation of daily rhythmicity compares favourably with that
of other important phenomena in the physical and biological worlds (Refinetti 2006).
Proper phase relationships among numerous physiological and behavioural
circadian rhythms, as well as between these rhythms and daily environmental cycles,
are crucial for the optimal health of the organism and its adaptation to the
environment (Pittendrigh 1965; Richardson 1990; Brock 1991). In particular, recent
chronobiological findings in horses were compared to what is known and highlighted
how the principles of circadian biology are applicable to equine husbandry and
veterinary care (Murphy 2010).
68 G. Piccione and C. Giannetto
The purpose of this article was to review the sparse but growing literature on
horses’ circadian rhythms and to highlight the importance of further research in this
field. The study of daily rhythms in the horse is of considerable importance in all
aspects of horse management to maintain the natural physiological behaviours of
this species, necessary to guarantee the welfare of breeding and athlete horses.
Maturation of the biological clock in foals
In several species of mammals, age-related changes in the circadian timing system
have been well documented. Typical changes include shortening of the circadian
period, alteration of the phase angle of entrainment to the L/D cycle, fragmentation
of the activity rhythm, decreased precision in onset of daily activity, altered rates of
re-entrainment following a shift in the L/D cycle and alterations in the response to
the phase-shifting effects of photic and nonphotic stimuli (Valentinuzzi et al. 1997;
Weinert 2000). The mammalian circadian system provides many potential targets on
which the ageing process could exert its effects. Ageing acts differently at several
different loci within the circadian system; its influence involves some but not all tissue
exhibiting circadian oscillations (Yamazaki et al. 2002).
Although developmental aspects of circadian rhythmicity have not received as
much attention as other aspects, some knowledge of the development of circadian
rhythms is available. In humans, newborns do not have daily rhythms of melatonin
secretion and body temperature that appearrespectively at three months of age and
at a year or more after birth (Kleitman et al. 1937; Abe and Fukui 1979; Attanasio
et al. 1986; Ardura et al. 2003). The homeostatic regulation of body temperature is
often incipient at birth and matures during the course of development. Maturation of
daily rhythms of body temperature are not observed until about 45 days of age in rats
(Allen and Kendall 1967); in newborns calves daily rhythms of body temperature
comparable with those of adults are not observed until two months after birth
(Piccione et al. 2003). In foals, monitored for 30 days after birth, the adult daily
pattern of body temperature oscillation was detectable within the first 10 days of life
(Figure 1) and was fully attained a month after birth (Piccione et al. 2002a). At birth,
the ability of foals to maintain thermal homeostasis is particularly limited and is
subordinate to the degree of maturity of the organism at the time of birth, to its
glycogen reserves and panniculus adiposus thickness. The existence of a rhythmic
pattern of body temperature, that emerges within the first 10 days of life, and matures
fully during the first month after birth, confirm the efficiency of thermal homeostasis
mechanisms in the newborn that working perfectly from the very first weeks of life
both in foals born within 340 days and those born between 341 and 355 days (Piccione
et al. 2005a). Different from that observed for the body temperature, blood pressure
circadian rhythm was not observed in foals (Figure 2) monitored for 40 days after
birth (Piccione et al. 2008a). It is probably due, not only to an incomplete anatomical-
functional development of the cardiovascular system (Piccione et al. 2005a), but also
to the immaturity of the autonomic nervous system, that with the circadian fluctuation
of endogenous opioids, hypothalamus-hypophysis axis, renin-angiotensin-aldosteron
complex, renal function and vasoactive peptides, affects the circadian rhythm of blood
pressure (Portaluppi et al. 1990, 1992, 1993, 1996). In the newborn, the heart rate is
forced to pump the blood through a vascular system that presents a strong elastic,
peripheral resistance and, since the newborn is not yet able to vary cardiac output, it
compensates the limited systolic volume by increasing the heart rate. The circadian
Biological Rhythm Research 69
rhythm of heart rate in newborn foals has not yet been investigated. Because blood
pressure and heart rate are correlated, and seeing that blood pressure in newborns no
showed a daily rhythm and the cardiovascular system is immature in this period of life,
we might suppose the lack of a daily rhythm of heart rate in foals, even if further study
is necessary to study this question in depth.
During the growth and development of organisms a high energy demand is
necessary, and the metabolism of young animals is characterised by a negative
energy balance (Hansel 1985). In foals during the first three months of life, distinct
disturbances in the acid–base balance was observed (Flisińska-Bojanowska et al.
1991), with high levels of lactic and pyruvic acids, transitory metabolites in processes
of energy freeing. The levels of pH, pO2, pCO2, lactic and pyruvic acids showed
circadian rhythms in growing foals, with acrophases similar to those of their mother
(Gill and Flisińska-Bojanowska 1994). The diurnal rhythm in lactic and pyruvic
Figure 1. Rectal temperature of newborn foals and their mothers, as well as ambient
temperature, recorded one hour before dusk and one hour before dawn for 30 days after
parturition (Reproduced with permission of Piccione et al. 2003).
70 G. Piccione and C. Giannetto
acids occurred in the third month of life, with acrophase at night hours (Flisińska-
Bojanowska et al. 1991). The presence of these rhythms underlay the high efficiency
of homeostatic mechanisms in these animals despite the action of strong
physiological factors as development of an organism.
Others indices of the energy supply to the growing organism are glucose and
cortisol. Circadian rhythms of glucose and insulin have been reported in horses
(Evans et al. 1974), but it was shown that these rhythms are lost when the animals
Figure 2. Systolic and diastolic blood pressure of newborn foals recorded one hour before
dawn and one hour before dusk for 40 days after birth. Symbols are means of the number of
animals indicated and the error bars indicate the standard errors of the mean (Reproduced
with permission of Piccione et al. 2008c).
Biological Rhythm Research 71
are fasted (Stull and Rodiek 1987), and modified when the animals are subjected to
different feeding regimes (Piccione et al. 2008b). In foals there is no circadian rhythm
of glucose (Flisińska-Bojanowska et al. 1989), probably due to the periodic suckling
during the day and night that does not allow the blood glucose concentration to reach
a daily peak. Circadian rhythms of cortisol have been reported in horses with highest
levels in the morning and the lowest in the evening (Hoffsis et al. 1970; Evans et al.
1974; Larsson et al. 1979); in foals a diurnal rhythm of cortisol level was found as
early as in the first week of life, its acrophase was observed at 04:30 and tends to reach
adult one at 7–11 weeks of life (Flisińska-Bojanowska et al. 1989, 1992). Gender
differences of peripheral steroid hormone concentration were observed in females
when compared to males and geldings (Fletcher et al. 2000). These hormones act on
the steroid receptors located on the osteoblastic cells, and are probably responsible
for differences in the osteocalcin serum level observed in male, female and gelding
mature horses (Giannetto et al. 2010). Mature and immature horses show different
bone metabolism influencing the daily rhythm of osteocalcin. The high rates of
skeletal modelling and remodelling during growth and the resulting greater variability
in bone marker concentrations determine the lack of a circadian rhythm.
Also, the behaviour of RBC, Hb and mean corpuscular haemoglobin (MCH) as
the indices engaged in the supply of oxygen to the growing organisms were
investigated. Higher values of RBC and Hb in foals than adults were observed. In
foals an increased action of erythropiesis was observed, erythrocytes produced and
released in peripheral circulation were immature and smaller than in adult horses.
The release of erythrocytes in foals, different from adult horses, is not circadian; it
may be a result of the action of not yet fully active erythropoietin in young foals
(Komosa et al. 1990).
The maturation of the central pacemaker and peripheral clocks were not observed
at the same time after birth in foals. The exact process that regulates maturation of
the synchronisation process is not completely understood and requires further study.
Daily rhythms of behaviour and physiological variables in horses
The circadian rhythm of body temperature is an especially important topic in
physiological research and involves the integration of efforts of two large groups of
researchers: those interested in the regulation of body temperature and those
interested in the mechanisms of biological timing. It took several centuries, however,
until systemic studies of body temperature were conducted, and it has been only in
the last 60 years that considerable attention has been given to daily variations in the
temperature of living organisms (Refinetti and Menaker 1992). Because of the
relative ease of monitoring body temperature and because of 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).
The thermoregulatory system is an encompassing system that utilises behavioural
and autonomic processes in integration with other physiological systems, such as the
respiratory, digestive, cardiovascular and motor systems (Piccione and Refinetti 2003).
Only in two studies conducted in horses, were measurements of body temperatureconducted often enough to allow a characterisation of the daily rhythm. Another three
studies were conducted to provide an estimate of the mean level of the body
temperature rhythm. The mean level in the five studies ranged between 37.7 and
38.48C. The daily range of excursion was found between 0.4 and 1.08C (Piccione and
Refinetti 2003).
72 G. Piccione and C. Giannetto
The body temperature of horses kept under natural photoperiods was found to
start its daily ascent at the time of sunrise to reach its acrophase 14 hours later,
during the scotophase. If the animals are kept under constant light neither the main
level nor the robustness of the rhythm differed from those of the animals kept under
natural photoperiods, underlining the endogenous nature of the body temperature
rhythm of horses (Piccione et al. 2002b).
In most studies where body temperature and locomotor activity were measured
simultaneously, the two 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 horses the first study to find a method for long-term registration of horse
behaviour was conducted by Gill (1991). He used an electronic apparatus for
continuous registration of human motor activity in hospital. Adaptation of the
device to the horse demands was not easy. It seemed that movements of the horse
neck and head are milder than those of human arms and hands. He had found a
circadian rhythm of motor activity in horses in several conditions, with different
patterns of activity from day to day due to weather, temperature, grazing and
abundance of flies. In later studies, an actigraphy-based data logger was used to
analyse the total activity of horses, which includes different behaviours such as
feeding, drinking, walking, grooming and small movements during sleep, indepen-
dent of the animal’s position, such as lying or standing. Actigraphs were placed by
means of headstalls that were accepted without any obvious disturbance, in
accordance with previous investigations showing that the behaviour parameters of
activity were correctly identified by collars (Berger 1993). Studies conduced on
Przewalski horses maintained under semi-reserve, on free-living Camargue and
domestically managed Dutch Warmblood foals showed that behavioural activities
occurred primarily during daylight (Boy and Duncan 1979; Berger et al. 1999;
Kurvers et al. 2006). In horses grazing behaviour occupies the majority of time and
consists of locomotor activity as well as feeding. The time spent grazing depends
on the structure and dispersion of patches on which animals prefer to feed, season,
age, sex and herbage availability (Houpt et al. 2001). Comparing horses housed in
standard stalls and horses kept in stalls with paddocks, locomotor activity showed
a diurnal pattern and the acrophase always occurred in the middle of the
photophase of the experimental photoperiod. In horses housed in stalls, locomotor
activity was prevalent during the diurnal period, while during the dark period there
were several activity peaks, mostly with lower intensity, and shorter than during
the light period, with several cycles of sleep. In horses kept in boxes with paddocks
the intensity of locomotor activity observed was lower than in animals housed in
stalls without a paddock, but constant in time; in these animals were also found to
have several cycles of sleep during the scotophase (Piccione et al. 2008c). As
previously observed, these activity bouts were of low intensity and ranged from 2
to 15 min during which horses lay down in lateral recumbency (Ruckebusch 1970).
Actograms, in any case, did not give exact information concerning the sleep of the
horse or whether it rests in a standing or lying down position (Gill 1991). It is
important to underline that the rhythmicity of locomotor activity in horses is
constant through the year (Figure 3); its acrophase persists in the middle of
scotophase during the four seasons, independently from the time of the sunrise
(Bertolucci et al. 2008).
A rhythm that phase leads another rhythm cannot be caused by it, unless the
phase lead is so great that it actually constitutes phase lag in the following cycle.
Biological Rhythm Research 73
Thus, in horses, the rhythm of rectal temperature (nocturnal rhythm) cannot be the
cause of the rhythm of locomotor activity (diurnal rhythm). Of course, the rhythm of
locomotor activity could be the cause of the temperature rhythm (Piccione et al.
2005b), but studies of human subjects in constant bed rest (Marotte and Timbal
1981; Kräuchi and Wirz-Justice 1994; Monk et al. 1996; Carrier and Monk 1997;
Murray et al. 2002) and correlated a studies in animals (Bolles et al. 1968; Honma
and Hiroshige 1978; Refinetti 1994, 1999; Gordon and Yang 1997) have clearly
shown that the temperature rhythm is autonomous, even if affected by the activity
rhythm. This means that either rectal temperature is connected to the circadian clock
by a different pathway than activity or it is controlled by a separate circadian clock
(Refinetti and Menaker 1992).
Figure 3. Total activity recorded in mares exposed to natural photoperiodic and
thermoperiodic conditions during vernal equinox, summer solstice, autumn equinox and
winter solstice. Each horizontal line is a record of one day’s activity, and consecutive days are
mounted one below the other. Total activity recorded during consecutive five-min periods is
indicated by vertical black markings. White and black bars at the top of the record indicate
photophases and scotophases (Reproduced with permission of Bertolucci et al. 2008).
74 G. Piccione and C. Giannetto
Another important factor that was taken into consideration in relation to the
circadian rhythm of body temperature is the breathing rate, even though they appear
to be phenomena running independently from each other. The circadian rhythm of
breathing rate was studied in horses housed in barns under 15.5/8.5 L/D cycle
(Piccione et al. 2005c). In these animals the acrophase of breathing rate anticipates
that of body temperature of nine hours. The factors responsible for the daily
oscillation of breathing remain unclear. It seems that neither the daily change
in metabolic rate nor that of body temperature are likely to be responsible for
the circadian pattern of breathing. In relation to the distribution of acrophase, the
rhythm of body temperature cannot be the cause of the rhythm of breathing, because
the acrophase of breathing anticipates the acrophase of body temperature. Also
the changes in locomotor activity have been dismissed as causative events, as the
oscillation of breathing was still present in humans subjected to prolonged bed rest
(Mortola 2004).
Heart rate and blood pressure also vary with changes in activity, posture and
other external stimuli. In horses, the electrical activity of the heart showed daily
rhythmicity. In particular daily fluctuations were observed in P–Q interval duration,
QRS duration, Q wave amplitude, T wave duration and amplitude, S–T segment
duration and Q–T duration (Piccione et al. 2005d).
Daily rhythms of haematological and haematochemical parameters in horses
More attention has been directed to the daily variations of the haematological and
haematochemical parameters (Figure 4). Some study of haematological parameters
in peripheral blood showing diurnal and seasonal changes have been performed in
horses (Gill and Rastawicka 1986; Gill et al. 1978; Komosa et al. 1990; Yashiki et al.
1995; Piccione et al. 2001). Studies carried out on thoroughbred and Arabian horses
showed the existence of a rhythmic pattern for some haematological parameters
which were not influenced by endogenous or exogenous factors (Gill et al. 1978; Gill
and Kownacka 1979). In thoroughbred horses, daily rhythms of haematological
parameters showed nocturnal acrophases. Red blood cell (RBC) and haemoglobin
(Hb) reached their peak immediately afterthe sunset, while haematocrit (PCV)
reached its peak some hours later. White blood cell (WBC) and platelets (PLT) did
not show any circadian rhythms (Piccione et al. 2005e).
Relating to the haematochemical parameters, in horses, daily rhythm was
observed in serum concentration of circulating fat soluble vitamins. They showed
similar diurnal acrophases. For vitamin A it was observed at 15:20, vitamin D
between 14:15 and 15:15, vitamin E at about 15:55 and vitamin K between 17:00 and
18:10 (Piccione et al. 2004a). A circadian pattern of fat soluble vitamins was
observed in humans and laboratory animals (Soulban et al. 1990; Lapenna et al.
1992; Kamali et al. 2001; Singh et al. 2001). However, little is known about the
temporal variability in serum vitamin concentration and its probable relationship to
the biological process, despite the important role of these substances in maintaining
the body’s physiological status.
In horses, the daily rhythm of vitamin K was studied, also in association with the
daily rhythm of clotting time parameters. Vitamin K showed its acrophase in the late
evening, as previously reported, while prothrombin time (PT) showed a nocturnal
acrophase between 20:08 and 02:08, activated partial prothrombin time (aPTT) and
fibrinogen no showed time-dependent variations (Piccione et al. 2005f). There are no
Biological Rhythm Research 75
other studies conducted in horses to compare these findings. Circadian variations of
blood clotting activity have been documented in several studies conducted on
humans, nocturnal rodents and rats (Everson 1960; Haus et al. 1990). In rats the
data observed by Pyörälä (1967) suggested that the levels of factors II, VII and X
tended to be lower during the active period than during the sleeping span. This could
be true also for horses that during their active period (daytime) showed the lowest
levels of PT, while during the night (their sleeping span) showed the PT acrophase.
For the first time the existence of daily rhythms of serum leptin in horses was
described by Piccione et al. (2004b). The profile of the serum leptin rhythms was
similar to those described in other mammalian species (Dallman et al. 1999;
Mastronardi et al. 2000; Kalsbeek et al. 2001); its lowest level was observed during
the photophase and the highest values were observed during the scotophase, and it
reached its acrophase at about six hours after sunset. Rhythmic leptin production is
Figure 4. Acrophases of the rhythms of 21 variables in horses. Circles indicate the means.
Horizontal lines indicate the 95% confidence intervals of the means (Reproduced with
permission of Picione et al. 2005a).
76 G. Piccione and C. Giannetto
regulated by the autonomic nervous system and is not influenced by feeding
schedules and exercise programs.
The eye and daily rhythms in horses
The mammalian eye, the main source of photic information to the central
pacemaker, is a remarkable rhythmic organ. It expresses circadian rhythms in
various processes at different levels of organisation from the molecular level, through
the cellular level, to the organ level and at the level of the visual system, including the
release of melatonin and dopamine, expression of visual pigment and visual
sensitivity (Nickla et al. 2002). Visual capabilities of the horse are inferior to the
human equivalents in terms of acuity and colour vision but are comparable in terms
of distance and depth perception and superior to humans’ under scotopic conditions
(Murphy 2010).
In horses daily rhythms of ocular parameters were found in the twomost diagnostic
parameters used in veterinary medicine to value eye diseases, tear production and
intraocular pressure (Piccione et al. 2008d; Bertolucci et al. 2009). Both parameters
showed a diurnal acrophase, the first nearly at the end of photophase, the second at the
end of daytime. For both parameters circadian rhythms persisted in constant darkness,
and were lost in constant light (Figure 5), also demonstrating that these temporal
variations in horses are generated by an endogenous circadian clock. Different from
the intraocular pressure, tear production of left and right eyes showed the same
trend, but was statistically different (Piccione et al. 2008d).
Exercise and daily rhythms in horses
Exercise imposes enormous perturbations on physiological systems, particularly
metabolic, circulatory, hormonal, and thermoregulatory mechanisms. Many
physiological and biochemical variables associated with exercise show circadian
rhythms. They oscillate almost in phase with each other (Drust et al. 2005; Mortola
2007), and the influence of the body clock on exercise performance could make the
difference between success and failure in a competitive contest (Reilly et al. 1997).
Evaluation of performance levels in the athlete is very complex, but can be simplified
by using individual functionality indicators. In the same way, the study of
chronophysiological response to physical activity in the athlete is very complex,
since it involves the whole organism. For example, maximal exercise of short
duration demonstrates circadian rhythmicity close in phase and shape to the core
temperature. This similarity applies to isometric force, anaerobic power and
anaerobic capacity, peak isokinetic torque, grip strength, joint flexibility and many
other measures (Reilly and Waterhouse 2009).
Therefore, the circadian rhythm of some physiological and hematochemical
parameters can influence the response to exercise and the adaptation of the organism
to the physical activity. These variables include rhythms of core temperature,
catecholamines, oxygen uptake (VO2), minute ventilation (VE) and heart rate (Reilly
and Brooks 1986). They are important for oxygen transport and utilisation, and
keeping blood gases constant throughout the 24 h (Mortola 2004; Piccione et al.
2004c). These oscillations could have an impact on exercise performance, since at the
peak of the daily oscillation in body temperature, the rightward shift of the
haemoglobin dissociation curve should favour transport of O2 to the peripheral
Biological Rhythm Research 77
tissues. Also, the greater resting values of breathing and heart rates might favour
pulmonary ventilation and cardiac output. On the other hand, the concomitant
greater levels of resting VO2 may offset these presumptive advantages. Piccione et al.
(2009a) have measured heart rate, blood pressure, rectal temperature, skin
temperature, blood glucose and lactate levels in horses performing an exercise
routine kept rigorously constant for eight days, in the morning or in the afternoon in
alternate order, with some days of rest after the first four days. Heart rate, blood
pressure and rectal temperature during the exercise routine differed between the
morning and the afternoon, all variables were greater in the afternoon, reflecting
the impact of endogenous circadian rhythms (Figures 6 and 7). At the impact of the
endogenous circadian pattern was added a constant value attained during exercise.
The differences in rectal temperature, blood pressure and heart rate remained
virtually unchanged during exercise between the morning and afternoon hours. Skin
temperature did not increase in the afternoon hours, suggesting that the peripheral
vasodilatation and heat loss remain unchanged. The circadian oscillations have
Figure 5. Daily rhythms of IOP (a) and STT (b) values in horses. Each point represents the
mean (+ SD) of the left or right eye. Grey indicates the dark phase of the 12/12 L/D period
(Reproduced with permission of Giannetto et al. 2009).
78 G. Piccione and C. Giannetto
additive effects on the absolute values of heart rate, blood pressure and rectal
temperature. This aspect seems relevant and should be taken into account in cases of
strenuous exercise in the afternoon because the safety margins against hyperthermia,
hypertension and pulmonary haemorrhage may be decreased. Physical exercise could
be an event regulating the daily rhythms of clotting parametersand platelet
aggregation in horse. It modified the acrophase of rhythm for 18:00–20:00 observed
in sedentary horses to 03:28–03:42 observed in athlete trained at 16:00 every day
(Casella et al. 2009). Therefore, the exact mechanisms that link exercise to platelet
aggregation and the events involved in exercise-induced platelet responses are not
completely understood.
Figure 6. Raw min-by-min data of heart rate (HR) for four horses in the morning (a.m.,
continuous line) and afternoon (p.m., dashed line), subjected to performance test. Each phase
of the test and its duration are indicated at the top. E1, E2, and E3. The difference among E1,
E2, and E3 was in the vertical height of the jumps, 1 6 0.8 m for E1, 1.15 6 1 m for E2, and
1.20 6 1.15 m for E3.
Biological Rhythm Research 79
Relating to the haematochemical parameters, an important role in athletic
performance is played by tryptophan and serotonin. The synthesis and metabolism
of serotonin in the brain increases in response to exercise (Chaouloff 1989). Its
increase in the brain is related to high levels of blood-borne tryptophan, the amino
acid precursor to serotonin. The rise of brain serotonin concentration is associated
with markers of central fatigue such as decreased motivation, lethargy, tiredness and
loss of motor coordination (Davis et al. 2000). The daily pattern regarding blood
levels of tryptophan and serotonin showed acrophases for both parameters during
the evening hours at the onset of the scotophase of the experimental photoperiod.
Tryptophan acrophase occurred 30 minutes earlier than serotonin acrophase. This is
inconsistent with the role of tryptophan hydroxylation in the control of serotonin
biosynthesis (Piccione et al. 2005g). Therefore, it is likely that exercise performed at
the time of the acrophase of the tryptophan rhythm affects the onset of physiological
fatigue, thus turning on the body’s exercise adaptation mechanisms in order to
maintain better physical performance.
In association with the physiological influence of circadian rhythm on
performance, it was observed that training acts as an exogenous synchroniser
capable of modifying and modulating the characteristic rhythmic features of the
main physiological parameters used for assessing the performance potential of the
athletic horse (Fazio et al. 2003). For example, the afternoon physical exercise of
horses might determine the shift of the homocysteine acrophase from diurnal to
Figure 7. Blood pressure (top left), body and skin temperatures (top right), blood glucose
(bottom left), and lactate (bottom right) for the six phases of the tests during the a.m. (black
columns) and p.m. (grey columns) hours. Symbols are mean values of the four horses, bars
indicate 1 SEM. *Statistically significant differences between a.m. and p.m.
80 G. Piccione and C. Giannetto
nocturnal (Fazio et al. 2006). Also non-esterified fatty acids (NEFA), palmitic,
stearic, oleic, linoleic and linolenic acids’ circadian rhythms are influenced by
physical exercise (Piccione et al. 2009b). A circadian rhythm of these acids was
observed in sedentary horse by Orme et al. (1994); NEFA showed their acrophases in
the morning at about 07:00, oleic acid between 04:00 and 10:00, while the acrophase
of stearic and linoleic acids occurred at 17:00. Subjecting the horse to a training
programme from 15:00 to 16:00 shifted NEFA and oleic acid acrophases to about
16:30. In the same hours it was also was possible to observe linoleic and linolenic
acids acrophases that did not differ from the acrophase observed in the sedentary
horse. In the athletic horse, the effect of physical exercise on the haematic
concentration of NEFA is well documented (Dunnett et al. 2002). NEFA are highly
energetic metabolites whose metabolic contributions are inversely proportional to
the work intensity and directly proportional to duration (Hambitzer and Bent 1988;
Caola and Assenza 2001). In sub-maximal exercise, NEFA, as an energetic substrate,
is needed in order to limit muscle glycogen consumption and delay the fatigue,
supporting long-term physical performance. Furthermore, the onset of physical
activity activates the sympatic nervous system that promotes lipomobilisation.
In horses, exercise and training are known to have also considerable effects on the
mechanisms of blood coagulation (Piccione et al. 2004d, 2005h), in particular the
effect of physical exercise on platelet aggregation in horses was observed (Johnstone
et al. 1991; Kingston et al. 1999). The exact mechanisms that link exercise to platelet
aggregation and the events involved in exercise-induced platelet responses are not
completely understood. Exercise was also able to influence the circadian rhythm of
platelet aggregation in a horse subjected to fitness training for 60 days, six days a
week (Piccione et al. 2008e). In this horse a shifting of acrophase from 19:00, the
value found in sedentary horses, to 03:40 was observed. Two weeks of rest were
enough to re-establish the acrophase value observed in sedentary horses.
Knowledge about time-of-day effects on the physiological responses to exercise in
horses, and the definition of the mechanisms involved in chronoperformance, can
have important implications on the planning of training schedules, working habits
and competitions.
Jet-lag and acclimatisation in athletic horses
The consequences of jet-lag for the equine athlete have become more relevant in
recent times owing to increased travel of performance horses across multiple time
zones for international competition.
Rapid displacement across multiple time zones results in a mismatch between the
previously entrained programme of the internal circadian clock and the new L/D
cycle, a phenomenon known as jet-lag (Winget et al. 1984; Nagano et al. 2003). In
human, shifts in the L/D cycle are known to cause malaise, appetite loss, sleep
disturbances, fatigue and reduced cognitive and physical performance, due to
decrease in reaction time, cardiorespiratory functions and muscle strength, until the
circadian clock system adjusts to the new environmental conditions, re-establishing
preferred phase relations among different rhythms and between these rhythms and
the external environment (Gander et al. 1985; Satoh et al. 2006). In horses, empirical
observations suggest adverse consequences after transport across different time
zones. However, the neuroendocrine mechanisms underlying these jet-lag effects
remain unclear, and the duration and severity of jet-lag related circadian disruption
Biological Rhythm Research 81
is presently unknown (Tortonese et al. 2003). The rate of adjustment to a new time
zone depends on the circadian output rhythm being measured, the number of time
zones crossed, the flight direction and the strength of the entraining cues in the new
time zone (Gander et al. 1985; Loat and Rhodes 1989). A comparison of equine
‘‘clock’’ genes with their human counterparts has revealed an unusually high
similarity between these two species at DNA level. Based on this similarity,
information on the effects of transmeridian travel derived from studies on human
performance can be used to provide guidelines to horse trainers (Maxwell 2004).
The first study to investigate re-entrainment of the melatonin and body
temperature circadian rhythm phase markers in the horse to an abrupt six hours
advance of the L/D cycle was conducted by Murphy et al. (2007). They found a rapid
adjustment of melatonin to the new photoperiod, completing the six hours phase
advance on the first post-shift day. In contrast, re-entrainment of the body temperature
rhythm was slower and not without disturbances in rhythm waveform. The acrophase
required three days to completely adjust to the new photoperiod. The transient
distortion of the body temperature rhythm post-shift was attributed to the phase
misalignment of multiple SCN, or other oscillators, which when in normal synchrony
contribute to a more robust body temperature rhythm waveform. The lower amplitude
of thebody temperature rhythm on the latter days post-shift suggested a weakening of
the association between the old zeitgeber and the body temperature circadian rhythm.
This weakening helps hasten the shifting of the clock to the new zeitgeber.
During transmeridian travel, associated with jet-lag, performance horses
travelling for international competition are subjected to extreme environmental
conditions, as observed in the occasion of the 1996 Olympic Games played in
Atlanta. The heat stress encountered by athletes during prolonged moderately
intense work, or when exposed to high heat and humidity, can be a critical factor,
ultimately limiting their ability to compete and often posing a serious risk to their
health. Exposure to high heat and humidity over a period of time provides an
opportunity for the body to adjust. Acclimatisation improves heat tolerance and
became effective when exercise is conducted in hot conditions that raise core
temperature and activate sweat glands providing improved protection against the
effects of heat stress (Lindinger et al. 1994; Reilly and Waterhouse 2009). When the
adaptations are acquired through acclimation protocols in heat chambers, there may
be a temporal component to the physiological adjustments, the adaptations being
more emphatic at the time of day the heat stress is imposed (Shido et al. 1999).
Environmental temperature has a similar effect to light on circadian organisation.
Changes in ambient temperature could influence the circadian rhythm of core
temperature either by threatening thermal homeostasis (which could be described as a
masking effect) or by acting directly as a zeitgeber that entrains the internal clock
(Holtzclaw 2001). The effects of changed temperature on circadian rhythms are very
difficult to test inmammalians in vivo since thermoregulatory reflexes restrict the rangeof
body temperatures to such an extent that the might have little consequence for the SCN
and circadian organisation (Reppert andWeaver 2001). Cable et al. (2007) argued that a
thermal stress that is presented repeatedly at a consistent time of daymay be anticipated,
enabling the thermoregulatory system to be activated in preparation for the environ-
mental challenge. In natural conditions, the adaptations are likely to be linked to the
prevailing ambient temperatures and the heat load imposed, irrespective of time of day.
Direct neural connections exist between the SCN and the hypothalamus responsible
for regulating body temperature rhythms (Lu et al. 2001), whereas messages from the
82 G. Piccione and C. Giannetto
SCN to the periphery are thought to occur via more circuitous routes, resulting in the
previously observed delayed re-adaptation of clock gene rhythms in peripheral organs
(Yamazaki et al. 2000).
Conclusion
Analysisof the current literatureonequines shows thatmanybiological processes exhibit
daily rhythmicity in horses. The acrophase of the principal parameters were observed
in the photophase, suggesting classification of the horse within the diurnal species.
In this species many rhythms are driven by an endogenous pacemaker located in
the SCN and are influenced by the photoperiod. For others, it is not clear if they are
strongly circadian, a by-product of human-imposed management regimes, and/or a
reflection of an equine ability to adapt to a range of differing environmental conditions.
The influence of external stimuli, such as exercise, on a circadian rhythm
endogenously generated is linked to the robustness of the rhythm. In fact, circadian
rhythms with a high percentage of robustness are not modified by external stimuli,
indeed themselves influence the response of the organism to the external stimulus,
while circadian rhythms with low robustness values change their acrophases in
relation to the time of external stimulus application.
Therefore the circadian rhythm of this species could be taken in consideration in
all aspects of horse management, starting from food administration, planning
feeding schedules that do not modify the natural physiological behaviour of this
species, continuing in the choice of living and working habits, planning of training
schedules and competitions, not only to obtain the better athletic performance, but
also to ensure the well-being in breeding practice.
Further studies involving experimental manipulation of external stimuli are
necessary to expand and to define the knowledge on the mechanisms involved in
chronophysiology, and to evaluate if the daily rhythms observed in athlete horses are
linked to the habitual time of training and have an influence on the response to
exercise carried out at different times of day.
Daily rhythms in horses have also been found to have clinical implications and
can influence disease occurrence or variation in severity over 24 hours, the response
of patients to diagnostic procedures and the effect of medical treatments. During
clinical examination the time of day of diagnostic parameters evaluation could be
useful to avoid errors in the sample interpretation, the management of disease and
drug administration. To optimise pharmaco therapies it is necessary to take into
consideration rhythm dependencies in the kinetics and dynamics of medications plus
predictable in-time variability in the manifestation and severity of diseases.
Acknowledgements
This work was supported by research grant PRIN 2008 (University of Messina, Italy).
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88 G. Piccione and C. Giannetto
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