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Pulmonary Function in the Exercising Horse
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Gas exchange is the main function of the lung, which ensures the transport of O2 from the air into the blood and of CO2 in the reverse direction.
In all studied animal species except the horse, the lung is able, when sound, to ensure adequate ventilation during exercise at any intensity. Conversely, horses experience hypoxemia, even at relatively low exercise intensities, as well as hypercapnia, during high intensity effort. These observations may seem surprising at first, given the numerous and substantial respiratory adjustments that occur in this species during exercise. However, some anatomic and physiologic peculiarities of the equine species may explain its inability to maintain adequate gas exchange during heavy exercise. The purpose of this paper is to discuss these equine respiratory system characteristics.
1. Morphologic Specificities and their Functional Consequences during Exercise
The main function of the airway is to carry air from the nose to the gas exchanging regions of the lung during inspiration and in the opposite direction during expiration. Equine airways are composed of the nostrils, the nasal cavities, the pharynx, the larynx, the trachea, the bronchi and the bronchioles. These structures are supported in some places by bones (nasal cavities) or cartilage (trachea and bronchi) and in other places by muscles only (nostrils, pharynx and larynx) or by very fine alveolar tissue (bronchioles) (Fig. 1). Although a reduction of cross-section diameter is potentially possible anywhere in the respiratory tree, the fact that some parts are unsupported by rigid structures makes them more susceptible to collapse than others. They consequently represent a potential bottleneck that may decrease the airway conductance.
Figure 1. A horse's respiratory tree with its support and musculature at different levels explaining why some parts are more susceptible than others to dynamic collapse during exercise.
The Horse’s Nostrils are large and mobile. The activation of their muscles allows expansion during inspiration, resulting in flaring of the nostrils and collapsing the nasal diverticulum, especially during exercise-induced hyperventilation. During expiration, nostrils are relaxed and flaccid. Therefore, in some horses expiratory flow may be noisy and the nasal resistance may increase to such an extent that it may induce exercise intolerance. In these animals, bilateral alar folds resection can abolish the respiratory noise and, in most cases, improves exercise tolerance .
Alar fold collapse and nostrils fluttering during effort may be also avoided by use of an external nasal support system (FLAIRtm strip), which is now commercially available. Because it stabilizes the nostrils, it most probably decreases their resistance to airflow. Its effect on performance and on exercise-induced pulmonary hemorrhage has been tested, but up to now, results are rather conflicting [2-5].
The nasal cavities provide a large surface area for heat and water exchange with their large turbinates and vascularisation. Autonomic regulation modulates vascular dilatation of sinuses, which in turn induces an increase or a decrease in nasal resistance. The sympathetic discharge occurring during exercise induces a vasoconstriction, which effectively enlarges the nasal cavities and decreases their resistance to airflow. This large vascular bed probably also plays a role in selective brain cooling of the horse during exercise, as shown by McConaghy et al., , who demonstrated that experimental upper respiratory bypass leads to an increase of the hypothalamic temperature.
The pharynx is divided into nasopharynx and oropharynx by the soft palate. Numerous lymphoid follicles are present in the mucous membranes of the dorsal and lateral walls of the nasopharynx and of the dorsal surface of the soft palate. The number and size of these follicles are particularly important in young horses and usually regress in mature horses. The question of knowing whether pharyngeal follicular hyperplasia may contribute to exercise intolerance is still controversial. Apparently, it has no effect on arterial blood gas tension during strenuous exercise, unless the lesions are extremely severe .
Similarly to the nostrils, the soft structures of the nasopharynx have no bony or cartilage support and therefore they tend to collapse during inspiration. This dynamic partial collapse is limited by tensor muscles contraction. This contraction, helps maintain upper airway patency, and seems to be partly regulated by reflexes. Their ascending pathway originates from mechanoreceptors and thermoreceptors located in the mucosa of the larynx and are stimulated by the increase of airflow . The descending motor response flows through the laryngeal branch of the vagus nerve. Paresis -secondary to any causes (e.g., inflammation of the region) - will impair the tensor efficiency.
The guttural pouches are paired diverticula of the Eustachian tubes which communicate with the pharynx via slit-like openings. These pouches contain vital structures like blood vessels (internal and external carotid arteries), cranial nerves (vagus, cervical sympathetic trunk, glossopharyngeal, hypoglossal and spinal accessory nerves) and retropharyngeal lymph nodes. Any infectious diseases of the guttural pouches may potentially damage these structures and consequently induce some dysfunction. For example, a lesion of the glossopharyngeal nerve and the vagus may induce a soft palate paresis and result in its dorsal displacement.
The physiological role of the guttural pouches remains open to debate. According to some authors, it could play an essential role in the physiology of swallowing . Recent work has provided strong evidence that it could also play a role as selective brain cooling, by allowing a decrease in the internal carotid artery temperature in horses, as they lack a carotid rete .
The equine larynx is a potential bottleneck in the upper airways. The additus laryngitis is delimited dorsally by the corniculate cartilages, laterally by the vocal folds and ventrally by the epiglottis (Fig. 2a). The larynx can vary from a tight seal, i.e., full adduction of the laryngeal structures during swallowing, to a maximal opening, i.e., full abduction of these structures during exercise-induced hyperpnea in order to decrease the resistance to airflow (Fig. 2b). The cricoarytenoideus dorsalis muscle is responsible for laryngeal abduction and vocal folds retraction.
Figure 2. Front view of the horse's larynx: a) resting horse; b) full abduction during exercise; c) asynchronism; d) hemiplegia.
Any impairment of this laryngeal opening due to structural reasons (e.g., rostral displacement of the palato-pharyngeal arch) or functional reasons (e.g., laryngeal hemiparesis) will be responsible for inadequate ventilation during heavy exercise  and will induce abnormal respiratory noises because of the increased airflow resistance  (Fig. 2c and Fig. 2d). The spectrum analysis of the resulting noise may even help to diagnose the pathology .
Idiopathic laryngeal hemiplegia is commonly encountered in horses, especially the tall ones with long necks. The larynx is innervated by the recurrent nerves. The left laryngeal nerve originates from the brain, travels down the neck into the chest as a part of the vagus. At the level of the heart, the recurrent nerve branches off the vagus and becomes an individual nerve, which has to travel back up the neck before it finally reaches the larynx. The high incidence of the idiopathic left hemiplegia in the horse (99% of the cases are left-sided) has been related to injuries due to the specific course of the left laryngeal nerve. Genetics factors also play a significant role .
Laryngeal function is generally graded from 1 (normal larynx) to 4 (hemiplegia). The intermediate grades, i.e., hemiparesis, are more difficult to identify and frequently necessitate endoscopy during exercise to accurately diagnose and discern the grades [15,16]. The functional deleterious impact of the laryngeal hemiparesis on blood gas tension and on performance has been demonstrated in numerous studies [11,16-18].
The larynx articulates into the intrapharyngeal ostium, which is an opening in the soft palate formed caudo-dorsally by the palatopharyngeal arch, laterally by the pillars of the soft palate and rostrally by the visible border of the soft palate. The laryngeal structures, i.e., the corniculate cartilages and the epiglottis, articulate with the ostium like a button in a "button hole" , forming an airtight seal when the horse breathes (Fig. 3a).
Figure 3. Lateral view of the pharynx-larynx region: a) physiological position during breathing; b) dorsal displacement of the soft palate. Note that in this case, the horse may breathe through the mouth, which is not the case in a).
This peculiar arrangement is specific to the equine species and explains why the horse is a compulsory nasal breather. Indeed, because of this anatomical characteristics, the horse is unable to switch from nasal to oro-nasal breathing when nasal resistance to airflow becomes too high, as occurs during exercise-induced hyperventilation.
In horses, the displacement of the caudal border of the soft palate to a position above the epiglottis (Fig. 3b), referred to as dorsal displacement of the soft palate, is not physiological, except when it occurs during swallowing, coughing or whinnying.
In all other conditions, this dorsal displacement is abnormal and will induce dyspnea, especially during strenuous exercise. It induces a narrowing of the upper airways and causes soft palate flapping, increase of upper airways resistance  and sometimes asphyxia in racing horses. Dorsal displacement of the soft palate is probably related to numerous etiological factors, among them, morphological and neuronal causes [21,22]. The etiopathology of laryngo-palatal dislocation is discussed thoroughly elsewhere in this book (see chapter by Derksen and Robinson) .
Despite its cartilaginous structure, the extrathoracic trachea is quite compliant and is susceptible to collapse during highly compressive transmural pressure which occurs during forced inspiration (Fig. 4) . Its compliance (and therefore its collapsibility) is, however, significantly decreased by smooth muscle contraction (due to the exercise-induced adrenal discharge) and by tracheal distension (due to head extension and neck stretching).
Figure 4. Relationship between the intra- and extra-airway pressures showing how the transmural pressure may be dilating or collapsing at different levels and at different times of the respiratory cycle.
After the bifurcation of the trachea into the right and left principal bronchi, the bronchial tree branches many times to the periphery of the lung via the primary bronchi, the segmental bronchi, the bronchioles and the terminal bronchioles. These intrathoracic conducting airways may collapse when the transmural pressure exerted on their wall are compressive. This is particularly true at the level of the small airways which do not have cartilaginous support. However, in contrast with extrathoracic airways where the partial dynamic collapse occurs during inspiration, the collapse of small airways occurs during forced expiration, i.e., when the extraluminal pressure is more positive than the intraluminal pressure (Fig. 4).
The Respiratory Muscles
In mammals the diaphragm, that separates the thorax from the abdomen, and the external intercostal muscles are the main inspiratory muscles. In horses, the serratus ventralis has an important role in assisting the inspiratory effort of the diaphragm, both at rest  and during exercise . On the other hand, the transversalis muscle is the principal active expiratory muscle. The intercostal muscles are also activated in the second part of inspiration and expiration .
The electromyographic study of respiratory muscle recruitment has been performed in healthy resting horses [25,27] in ponies during hypoxia  and in horses during exercise . Electrical activation of the diaphragm as well as its mechanical output increases linearly with exercise intensity .
The activity of the ventilatory muscles in ponies has been indirectly estimated by the increase in the muscles perfusion [26,30,31]. The ventilatory muscles comprise 5.5% of the total body weight and receive 10% and 15% of the cardiac output at rest and at maximal exercise, respectively. During exercise, the costal diaphragm blood flow exceeds the blood flow to all other inspiratory or expiratory muscles: It increases over 20-fold in maximally exercised ponies. Moreover, during maximal exercise, diaphragmatic blood flow reaches its upper limit [32,33]. Adenosine infusion, which causes a marked vasodilatation in the pony's diaphragm at rest, fails to elicit any further vasodilatation, indicating that, in this breed, vasodilator capacity is completely utilized in the pony during exercise . This suggests a potential limiting factor of the ventilatory machinery during heavy exercise.
Sternothyroideus and sternohyoideus muscles contraction during exercise retracts the hyoid apparatus and the larynx caudally. Therefore, they help to improve or to maintain upper airway patency and stability in normal horses and may be considered as respiratory muscles . It has been shown that their transection, frequently used to prevent recurrent dorsal displacement of the soft palate in race horses, alters upper airway mechanics in healthy horses during intense exercise by facilitating pharyngeal collapse .
Last, the abdominal muscles (external and oblique abdominis, transverse and rectus abdominis, transversus thoracis) and the internal intercostal muscles are expiratory muscles. When they contract, they increase the abdominal pressure, forcing the relaxed diaphragm forward and reducing the thoracic volume, a breathing strategy that, contrarily to the other species, is physiological in horses .
2. Peculiarities of Respiration During Exercise
The main factors involved in gas exchange are ventilation (i.e., how air gets to the alveoli), perfusion (i.e., removal of gas from the lungs by the blood), ventilation-perfusion ratio (i.e., how matching of air and blood distribution in the lung influences the gas exchange), diffusion (i.e., how gas gets across the air-blood barrier), gas transport (i.e., how gases are moved from lungs to the tissues), mechanics of breathing (i.e., how the lungs are moved) and control of breathing (i.e., how gas exchange is adjusted to the metabolic demand).
Exercise imposes a potent stress on the ventilatory pump: As speed increases, minute ventilation (i.e., tidal volume times breathing frequency) increases almost linearly. Expired minute ventilation, which averages 80 L/min at rest, may reach values in the vicinity of 1800 L/min during heavy exercise [35,36]. The relative contributions of increases in tidal volume and respiratory frequency depends on the gait of the animal.
At the walk and trot, respiratory and step frequencies are independent, although studies have reported that sometimes they may be coupled. However, this coupling is neither constant nor compulsory [37-39], but when it occurs, it seems that the "abdominal piston" acts in synergy with the respiratory pump  probably reducing the cost of breathing.
In Standardbred horses trotting at 10 m/sec with a 6% slope, a tidal volume of about 18 L and respiratory frequency of 87 breaths/min have been reported . This breathing strategy is different from that observed in Thoroughbred horses at similar exercise intensities, but despite the lack of locomotion/respiration coupling, Standardbred horses did not reach higher expired minute ventilation than Thoroughbred do and they were hypoxemic and hypercapnic, just as Thoroughbreds are [40,41].
Once the horse gallops, there is a compulsory linkage between step and respiratory rates [38,42-44]. Step and respiratory frequencies average 110 to 130 /min, with maximum values of 148/min [45,46]. Tidal volume between 12 and 15 L have been reported in fast galloping horses [35,46-48].
The mechanisms underlying the coupling, as well as its physiological significance are not yet well understood. It is probably due to a mechanical linkage with the visceral content acting as a piston, and flexion of the back and loading of the thorax by the forelimbs . During protraction of the forelimbs, the rib cage is pulled forwards and outwards, favouring inhalation (Fig. 5a). During weight-bearing, the rib cage absorbs forces and is compressed, favouring exhalation (Fig. 5b).
Figure 5. Respiration-locomotion coupling in galloping horse. a) Inspiration: Protraction of the forelimbs, caudal displacement of the visceral piston, lumbosacral extension, cervical movement which pulls the rib cage forwards and outwards; b) Expiration: Weight bearing, down movement of the neck and cranial displacement of the visceral piston that compress the rib cage, lumbosacral flexion.
Experimental evidence shows that, in galloping horses, back flexion rather than the visceral piston mechanism assists breathing . The mechanism could consequently give a mechanical advantage to respiration. These conclusions have nevertheless been challenged by a work showing no evidence that diaphragmatic electrical activity or mechanical output was "spared" as horses ran at progressively increasing intensity: Total diaphragmatic electrical activity and mechanical output were proportional to exercise hyperpnea independent of the existence of a coupling between gait and respiration .
It must be emphasized that this coupling is not absolute in healthy horses. Indeed, galloping horses, either on the track or on the treadmill, may sporadically show a "big breath" which continues for two or three strides . An extreme value of 29.7 L tidal volume has been observed in a horse decoupling the 1:1 step/respiratory frequency ratio for a 2:1 ratio . The reason for this breathing strategy remains to be investigated but these horses seem to run better after this lung hyperinflation.
Although frequently suggested, a relationship between stride length and tidal volume amplitude has not been demonstrated so far .
Last, breathing patterns during exercise may be altered by respiratory troubles. Qualitative and quantitative assessments of respiratory airflow, pattern of breathing and tidal breathing flow-volume loops at rest and during exercise may, in some cases, help to diagnose clinical and subclinical obstructive respiratory diseases [52-55].
Only a part of the inspired volume reaches the area of the lung where gas exchange takes place: This is the alveolar ventilation. The remaining part of the minute ventilation is wasted in the regions of lung where no gas exchange occurs. This is the physiological dead space ventilation. It includes the conducting airways (anatomical dead space) and the alveoli which are ventilated but not perfused (alveolar dead space). The dead space to tidal volume ratio averages 50 - 60% in the resting horse [56,57], a percentage twice as large as reported in other athletic species like man and dog.
Exercise-induced changes in alveolar ventilation and dead space to tidal volume ratio in horses depend on the type of exercise performed [47,58-61]. During mild to moderate exercise, the dead space volume does not change significantly . Therefore, the increase in tidal volume will increase the alveolar ventilation and decrease the dead space to tidal volume ratio.
If the exercise is prolonged at a constant rate, the dead space ventilation will increase by a simultaneous increase in respiratory frequency and in the dead space to tidal volume ratio [59,60]. This adaptation probably reflects the thermoregulatory role of the respiratory system .
Last, during intense effort, there is a decrease of the same ratio from about 60% to 20% [57,60]. In absolute terms, the physiological dead space is reduced from 3.5 L at rest to 2.5 L during heavy exercise. Because the anatomical dead space averages 2.5 L at rest  and is expected to remain unchanged during exercise, the exercise-induced difference in the dead space (1 L) is probably attributable to the disappearance of the alveolar dead space (i.e., alveoli which are ventilated but not perfused) induced by the recruitment of previously non-functional pulmonary capillaries .
The distribution of ventilation is not uniform in the lung, even in healthy horses. This is for two different reasons. The main one is that the intrapleural pressure changes are not uniform all over the thoracic cage . A second reason may be the occurrence of some inequalities in regional small airways resistance and/or alveoli compliance: Inhaled air preferentially enters the areas of the lungs with low resistive airways and highly compliant alveoli (Fig. 6).
Figure 6. The "Otis model" shows how an increase in bronchial resistance leads to ventilatory asynchronism and eventually to impairment of gas exchange, especially when respiratory rate is high.
The resulting ventilatory asynchronism is moderate in healthy horses and does not have significant effects on gas exchange at low respiratory frequencies. However, in horses with significant asynchronism (i.e., subclinical small airway disease) this phenomenon will significantly impair gas exchange and may result in poor performance (Fig. 6). Moreover, exercise, by increasing the respiratory frequency, probably also magnifies the regional differences in ventilation. The lobules which have a long time constant for filling do not fill adequately before expiration begins. Consequently ventilation/perfusion mismatching and hypoxemia result.
Two peculiarities of the horse’s lung may nevertheless partially compensate the non-uniformity of ventilation: (1) the interdependence between adjacent lung regions which is the result of the intricate mesh of interconnecting elastic and collagenous tissue fibers in the lung  and (2) the collateral ventilation between adjacent lung areas. However, in the horse, these collateral pathways, due to their high resistance to airflow, are of limited usefulness at low respiratory frequencies and probably of no functional importance at all at high respiratory frequencies .
On the other hand, the interdependence between adjacent lung areas may induce abnormal stresses on some lobules. Indeed, with increased airflow resistance and/or decreased compliance, the tissues are stretched and compressed by the surrounding lung parts . It has even been suggested that exercise-induced pulmonary hemorrhage could sometimes be a consequence of this pulmonary overstretching .
The lung is perfused with blood from the pulmonary and bronchial circulations. The pulmonary circulation receives the total cardiac output from the right heart. The branches of the pulmonary artery carry mixed venous blood to the lung, and accompany the bronchi and form rich capillary plexuses on the walls of the alveoli. Here the blood is arterialized and returned to the left heart by the pulmonary veins.
In man, blood flow distribution is mainly influenced by gravity and shows consequently a vertical gradient according to the relative magnitude of pulmonary arterial, venous and alveolar pressures [66,67]. The ventral regions receive more perfusion per unit lung volume than the dorsal regions, and blood flow in the lung can be divided into 3 or 4 zones (Fig. 7a).
Figure 7. Old and new concepts of pulmonary blood distribution in the equine lung. It was previously thought that gravity was the main determinant in lung blood perfusion and resulted in a vertical perfusion gradient (a); however, it has recently been shown that blood is preferentially distributed in the dorsal parts of the lung, resulting in a caudo-dorsal gradient.
It has been long thought that the same was true for large quadrupeds, but recent works indicated that, in these animals, even at rest, gravity is a minor factor determining blood flow distribution, despite the large hydrostatic gradient imposed by the height of the lung. Blood flow is preferentially distributed in the dorsal regions of the lung  (Fig. 7b), a phenomenon which is exacerbated during exercise .
The greatest perfusion redistribution occurs at light exercise intensity, with little changes occurring when intensity further increases. The dorsal blood redistribution during exercise is still exacerbated (Fig. 8) and more heterogenous in horses with heaves than in healthy ones .
Figure 8. Computed relationship between exercising and resting pulmonary perfusion. Scintigraphic images in a healthy horse showing preferential blood redistribution to the caudo-dorsal region (red areas; courtesy of D. Votion).
Moreover, there is a considerable heterogeneity in each isogravitational plane  which obviously is in disagreement with the gravitational model. More than 70% of variations of pulmonary blood flow at rest and during exercise seems consequently to be determined by a fixed spatial pattern that is most likely related to the structural anatomy of the pulmonary vessel tree. Accordingly, despite a redistribution of pulmonary blood flow, heterogeneity of perfusion remains during effort . Furosemide, which is frequently used for its putative preventive effect on hemorrhage in susceptible horses, slightly attenuates this blood redistribution, but does not influence flow heterogeneity . The remaining 30% variation of blood distribution may be attributable to modulation of vascular diameter and probably other factors, which remain to be discovered.
Vascular diameter is modulated by smooth muscles in arteries and veins, that respond to vasoactive compounds. In horses, nitric oxide has vasodilatory effects and an effect on the pulmonary circulation during exercise has been strongly suggested by several experiments using systemic administration of nitric oxide or nitric oxide inhibitor [72-74], while not confirmed by another experiment where oral administration was used  nor by Manohar and Goetz .
The magnitude and mechanism of the vascular smooth muscle responses depends on numerous factors such as the pre-existing level of muscle tone and the integrity of the pulmonary vascular endothelium [77,78]. Indeed, endothelial cells are known controllers of vascular smooth muscle tension. For example, endothelin, a potent vasoactive endogenous peptide, exerts a vasoconstrictive action on pulmonary circulation in horses . On the other hand, endothelial cells mediate flow-dependent vasodilatation by increased production or release of their NO or other endothelial derived relaxing factors [80,81]?
In horses, Pelletier et al., [78,82] demonstrated that the vasodilator response of the pulmonary vessels was different according to their distribution from top to bottom. Methacholine induced a relaxation of the top vessels while it had the opposite effect on the bottom ones. This regional difference in endothelium-dependent response and vascular reactivity is now put forward to explain the preferential location for occurrence of pulmonary hemorrhages: Dorsal pulmonary arteries probably dilate more during exercise that the ventral ones and are consequently better perfused. The authors suggested that differences in nitric oxide release in response to differences in blood flow increase could be partly responsible for this observation.
A modest autonomic innervation, with both adrenergic and cholinergic components, is found in the muscular vessels of the pulmonary circulation. Stimulation of the sympathetic nervous system constricts the blood vessels of the lung, whereas parasympathetic stimulation causes vasodilatation.
Lung volume also plays a role in pulmonary blood flow by modulating vascular resistance. A distinction must be made between extra-alveolar vessels (pulmonary artery, arterioles, venules and veins) and alveolar vessels (that are the thin-walled capillaries which perfuse alveoli). The cross-section of the first ones are dependent on the pressure changes in the interstitial tissues rather than in the alveoli, while the opposite is true for the second ones. Consequently, vascular resistance varies with degree of lung inflation as described in Fig. 9.
Figure 9. The vascular resistance curve depends on the degree of lung inflation. At residual volume, extra-alveolar vessels have a low diameter and alveolar vessels, a large diameter. Conversely, at total lung capacity, extra-alveolar vessels are distended and alveolar vessels are narrowed. In both cases, pulmonary vascular resistance is increased. It is minimal between these extremes, i.e., at functional residual capacity.
Last, the modification of ventilation in some regions of the lung or in the whole lung also influences pulmonary perfusion. In unventilated regions of the lung, alveolar hypoxia occurs, inducing local hypoxic vasoconstriction. This constriction provides a mechanism to redistribute pulmonary blood flow from less ventilated to well ventilated regions and therefore improves the ventilation/perfusion ratio and the gas exchange.
During strenuous exercise, pulmonary blood flow increases approximately 8-fold [83-87]. The pulmonary right-to-left shunt of the cardiac output that is approximately 1% at rest may decrease up to 0.4% during heavy exercise .
A marked simultaneous pulmonary hypertension is a unique feature of exertion in horses and ponies: The mean pulmonary arterial pressure rises about 3-fold, from 28 mmHg at rest to about 84 mmHg at a fast gallop, with maximal reported values of 100 mmHg [88-95]. As well, pulmonary capillary, wedge and venous pressure significantly increase [76,96]. The factors that increase vascular pressure in the horse are not clear. A very high left atrial pressure that is necessary for rapid ventricular filling in this species where heart rate may increase up to 240 beats per minute could be partly incriminated.
This elevation in pulmonary pressure is nowadays largely thought as being one of the major factors responsible for the occurrence of pulmonary hemorrhages during strenuous exercise. While some authors did not find a relationship between pulmonary vascular pressure and occurrence of blood into the airways after exercise , others identified a pressure threshold of 90 mmHg above which hemorrhages occur in the lungs .
Pulmonary vascular resistance decreases with exercise . Actually several factors should increase the vascular resistance, such as the compression of the extra-alveolar lung vessels during forced expiration (small lung volume) or alveolar vessels during forced inspiration (lung distended)  (Fig. 9). The increase in blood viscosity related to the exercise-induced rise in packed cell volume could also influence the vascular resistance: Although an in vitro study has shown that blood viscosity is not a major factor contributing to vascular resistance in horses , it seems that, in humans, each increase of 1% in the packed cell volume induces an increase of 4% in the pulmonary vascular resistance . On the other hand splenectomized exercising ponies had a lesser increase in pulmonary arterial pressure when compared to normal ones . Other factors should decrease resistance: (1) dilatation (increase of their cross-sectional area and consequently decrease of their flow resistance) and (2) recruitment of the vascular bed . In horses during exercise, the final result of these opposite effects is an approximate 2-fold decrease of vascular resistance (approximately from 0.14 to 0.06 mmHg/ml/min/kg) with strenuous exercise .
The use of the arterial occlusion technique has allowed estimation of the pulmonary capillary pressure in exercising horses. Study of the relationship between pulmonary capillary pressure and the arterial and wedge pressures suggested that the major component of pulmonary vascular resistance is the pre-capillary sphincter (arterial capillaries). The post-capillary resistance (venous capillaries) is, conversely very low. This physiological adjustment consequently offers protection against large increase in pressure at the level of the capillaries .
The great increase in pulmonary arterial pressure and the consistent exercise-related hypoxemia has led to the suggestion that diffusion is altered by accumulation of interstitial water and occurrence of interstitial edema. In human beings and pigs, this phenomenon exists leading to mismatching of the ventilation/perfusion ratio [103,104]. A recent experiment demonstrated that there is no interstitial water accumulation in exercising horses , which is entirely compatible with the preservation of ventilation/perfusion ratio previously reported . Why hypertension and the increase in the rate of fluid filtration across the capillary walls of the lung do not result in pulmonary edema may be explained by the fact that in horses, as in other species , the lymphatic system has a substantial capacity to drain the pulmonary interstitial space [87,100]. Moreover, the "pumping" action associated with the large and frequent pressure changes in the equine thoracic cage during exercise could contribute to the efficiency of the lymphatic drainage.
The bronchial circulation receives approximately 1 - 2% of the cardiac output from the left heart. It supplies the airways, large pulmonary blood vessels, septae, pleurae and other lung structures. At the level of the terminal bronchioles, pulmonary and bronchial circulations anastomose. Most of these anastomoses occur at the level of the capillaries and veins rather than the arteries [106,107]. These post-pulmonary shunts may theoretically be partly responsible for the fall in the arterial O2 partial pressure observed during exercise. However, a shunt of 1%, which is a reasonable approximation, would reduce arterial O2 partial pressure by 5 mmHg, while the actual decrease during exercise is much larger .
When pulmonary perfusion is locally impaired, the bronchial circulation may proliferate and maintain some blood flow throughout the lung, thus contributing partly to gas exchange . Such proliferations also may occur during pulmonary inflammation. Because they are extensive in the dorso-caudal regions of the lungs, which are the sections that sometimes bleed during exercise, this neovascularization has been related to the occurrence of exercise-induced pulmonary hemorrhage . Pulmonary and/or bronchial hypertensions are strongly suspected to also contribute to exercise-induced pulmonary hemorrhage, but this has not been actually demonstrated as yet.
Ventilation/Perfusion Matching - Gas exchange ultimately depends on the matching of ventilation and perfusion. It is efficient in lung regions where the ratio is 0.8 to 1.0 - 1.2, which is the case for most regions. However, regions exist that are excessively perfused, i.e., ventilation/perfusion ratio < 0.8, or ventilated, i.e., ventilation/perfusion ratio > 1.2. [110-111].
In horses, the ventilation/perfusion ratio is not influenced by gravity, i.e., it is uniform from the top to the bottom of the lung, suggesting that in this species the gradient in lung ventilation is matched by the gradient in lung perfusion [66,110,111].
In heavily trotting and galloping horses, there is only a slight mismatch of ventilation and perfusion [41,88,112]. This physiological adaptation contrasts with what is observed in humans and pigs, where the ventilation/perfusion ratio is non-uniform at rest and a true mismatching occurs during exercise [103,104,113]. The mechanism of this light exercise-induced ventilation/perfusion inequality in horses remains to be established but it seems that the hypothesis that occurrence of an interstitial edema as it is most probably the case in man  may be ruled out in the equine species . This mismatch accounts for 25%  to 40%  of the increase in the alveolar-arterial pressure difference in O2.
In subjects suffering from airway disease, exercise will enhance ventilation/perfusion mismatching, by impairing either ventilation or perfusion, or both. In the horse, the collateral ventilation has little ability to compensate for the non-permeability of small airways . Therefore, with respiratory disease, some lobules will take a longer time to expand, leading to an inadequate ventilation and perfusion ratio, and hypoxemia and sometimes hypercapnia will result. Recent studies have also shown that gas exchange impairment during exercise may also result from inappropriate perfusion redistribution in chronic obstructive pulmonary disease (COPD) horses  and in Standardbred horses with red cell hypervolemia .
Diffusion is the passive process whereby O2 passes from alveoli to capillary blood and CO2 passes in the reverse direction.
Once the horse runs and crosses the threshold of a 60% of VO2max, arterial hypoxemia and hemoglobin desaturation occurs [86,115]. The alveolar-arterial O2 tension difference, which averages 4 mmHg at rest, widens and may reach values as high as 30 mmHg  suggesting diffusion impairment.
Classically, hypoxemia is primarily related to one of the following factors: (1) decrease in the partial pressure of O2 in the inspired air, (2) right to left vascular shunts, (3) ventilation/perfusion mismatching, (4) diffusion impairment, (5) alveolar hypoventilation.
Right-to-left shunts and mismatching of ventilation and perfusion have been shown to be accessory factors in producing hypoxemia during exercise . A mild ventilation/perfusion ratio inequality accounts for 25 to 40% of the widening of the alveolar-arterial O2 tension difference. Alveolar-capillary diffusion impairment, and less important alveolar hypoventilation, seem to be responsible for the remaining 60 to 75% of this widening.
Actually, during heavy exercise, different and opposite factors may influence the diffusion capacity.
On the one hand, several factors should improve diffusion. First, a mixed venous O2 pressure as low as 16mmHg has been reported during a fast gallop . Although the alveolar O2 pressure decreases, the pressure gradient between the capillary blood and the alveolar air is increased, improving O2 extraction and diffusion. Second, the increased blood flow results in functional new alveoli previously unperfused at rest, and therefore increases the functional alveolar surface. Third, the dramatic increase of hemoglobinemia promotes O2 diffusion by increasing the number of binding sites available.
On the other hand, despite the 50 - 60% increase in the volume of the pulmonary vasculature (due to the recruitment and enlargement of the capillary bed), the 8-fold increase in cardiac output leads to a dramatic shortening of the capillary transit time and consequently to a decrease of the time for O2 equilibration, and thus, diffusion impairment  (Fig. 10).
Figure 10. Schematic view of O2 diffusion across the alveolar-capillary membrane at rest (green line) and during exercise (orange line). The capillary transit time is reduced to about 250 msec in the latter condition. As a consequence, complete equilibration does not occur before blood leaves the gas exchange area. (A: alveolar; v: mixed venous blood; a: arterialized blood).
Other athletic species have a shortened transit time during exercise (0.29 sec in the dog, 0.35 sec in the pony and 0.5 sec in man) but neither the dog nor the pony demonstrate diffusion limitation during exercise . Therefore, it may be hypothesized that these species are able to compensate for their short capillary transit time with other physiological adaptations, which are either lacking or inadequate in the horse.
Last, hypoventilation also plays a role in the occurrence of hypoxemia during exercise as demonstrated by the fact that the widening of the alveolo-arterial gradient in O2 is diminished when a helium-oxygen mixture is substituted for air .
Blood Gas Transport
The oxygen content of blood is mainly determined by the hemoglobin concentration and its saturation with O2. When hemoglobin is saturated with O2, 100 ml of blood carries about 20 ml of O2, including the 0.3 ml of O2 dissolved per 100 ml of plasma.
The saturation of hemoglobin depends on the arterial O2 partial pressure which depends in turn on the amount of O2 dissolved in the plasma. Above an O2 partial pressure of approximately 70 mmHg, the oxyhemoglobin curve is flat and any increase in partial pressure will add little O2 to hemoglobin: i.e., the hemoglobin is nearly saturated and only a few (3 - 5%) of the binding sites are still available. Below an O2 partial pressure of 60 mmHg, the oxyhemoglobin curve shows a sharp decreasing slope. At the partial pressure encountered in the tissues (about 40 mmHg), blood loses about 25% of its O2 to the tissues. When the metabolism is high, i.e., during exercise, the tissue partial pressure of O2 is further reduced and more O2 will be released. Arterial O2 partial pressure in blood returning from all tissues averages 16 mmHg during heavy exercise. It may consequently be assumed that O2 tension at the level of the exercising muscle is much lower and this increases the driving pressure for O2 diffusion at this level. Last, increases in blood temperature, [H+], partial pressure of CO2 and intracellular concentration of certain organic phosphates (2,3 DPG) induce a right shift of the curve , promoting a higher O2 release at the level of the metabolizing tissues (Fig. 11).
Figure 11. Oxyhemoglobin dissociation curve at rest (blue curve) and during exercise (red curve) showing the right shift related to the decrease in pH and the increase in temperature and PCO2. Such a shift facilitates dissociation of O2 from Hb and consequently the release of O2 to the tissues. This effect is more remarkable in the tissues (PO2: 40 mmHg) than at the level of the lung (PO2: 100 mmHg).
When the number of erythrocytes is low and therefore when hemoglobin is reduced, as in anemia, the O2 content is reduced, despite normal arterial O2 partial pressure and hemoglobin saturation. On the contrary, when the packed cell volume increases, as during exercise in horses, the O2 content increases, even when the arterial O2 partial pressure is reduced. The increase in the packed cell volume and the consequent increase in hemoglobin due to splenic contraction is an adaptation to exercise specific to the horse (and the dog), providing almost 50 - 60% more binding sites for O2 during exercise . This represents an adjustment which partly compensates for the fall in arterial O2 partial pressure and hemoglobin desaturation. It has been hypothesised that too great an increase in packed cell volume may be disadvantageous from a hemodynamic point of view, because it increases the blood viscosity . However, this theory has been rejected by an in vitro study showing that viscosity is not a major factor contributing to vascular resistance, because actually the apparent viscosity of blood decreases when shear rates increases .
Recent works have nevertheless failed to demonstrate a deleterious effect of phlebotomy or a favourable effect of blood transfusion on both oxygen consumption and performance .
Carbon dioxide results from metabolic processes occurring in the tissues and once produced, it diffuses from the cells into the capillary blood. At high intensities of exercise, i.e., 100% of VO2 max, the large muscular mass produces an extraordinary amount of CO2 and the lung seems unable to completely eliminate it. The development of a relative CO2 retention in horses, even when running without a mask, is unique among mammals . Human athletes develop a compensatory hyperventilation during heavy exercise to ensure high alveolar O2 partial pressure, which in turn hastens the rate of equilibrium of alveolar gases with mixed venous blood and also provokes a decrease in the arterial partial pressure of CO2 to about 30 mmHg . Obviously, in exercising horses, there is a lack of truly compensatory hyperventilation and this contributes to the development of exercise-induced hypercapnia .
Mechanics of Breathing
Volume changes in the respiratory apparatus imply that work is being performed on the respiratory system, mainly expanding or compressing the gas in the lungs, and displacing it in and out of the airways. The driving forces exerted by the respiratory muscles are opposed mainly by static forces (elastic, gravitational and surface) and flow resistive forces (viscous and turbulent resistance of the gases and viscous resistance of the tissues). Inertial forces, negligible at rest, are also of importance in running horses.
The study of the relationship between the pressures exerted on the respiratory system (which are the causes) and the changes in volume and airflow that result (which are the effects) is the basis of the mechanics of breathing (Fig. 12).
Figure 12. Mechanics of breathing measurement: Pleural pressure is measured by the esophageal balloon technique and respiratory airflow is obtained using a pneumotachograph. Tidal volume is derived by integrating the respiratory airflow signal, and several mechanical parameters are calculated from standard formulae by a computer.
Because the visceral and parietal pleura are maintained in close apposition, the lung and the thorax interact mechanically. During inspiration, the intrapleural pressure (which at rest and in quiet conditions is subatmospheric) decreases, while during expiration, it increases towards its value at functional residual capacity, i.e., lung volume before inspiration.
Once exercise starts, the maximal pleural pressure changes increase and in Thoroughbred horses during maximal exercise, changes as high as 8.5 kPa or 80 cm H2O have been recorded . In this condition, respiratory frequency is about 120/min and, therefore, such a pressure swing takes less than 250 msec. Because the transformation of chemical energy into mechanical energy has a finite rate, the force-speed characteristics of the respiratory muscles could be a limitation to the ability of ventilation to increase during strenuous exercise.
The total pulmonary resistance quantifies the receptivity of the airways to airflow. The radius of the airway is of critical importance in determining the magnitude of this resistance to flow: When it is divided by two, resistance is increased by 16-fold.
During quiet breathing, 50% of the total pulmonary resistance results from the nasal passages, 30% from the remaining upper airways and 20% from the intrathoracic airways (Fig. 13) .
Figure 13. Relative contribution of nasal (dark blue area), laryngeal + extrathoracic tracheal (middle blue area) and lower airway (light blue) resistances to total pulmonary resistance at rest and at a fast trot (A: total resistance, B: inspiratory resistance; C: expiratory resistance) .
The importance of the relative contribution of the nasal cavities is not specific to horses but this species, in contrast to others, cannot switch from nasal to oronasal breathing.
While not significantly modifying the relative contribution of each part of the respiratory tract to the total pulmonary resistance, strenuous exercise induces a substantial 2-fold increase in this resistance [35,124]. Actually, total pulmonary resistance is the result of two kinds of opposed factors: Physiological ones which tend to decrease the resistance and physical ones which tend to increase it. During exercise, dilatation of the external nares, full abduction of the larynx and bronchodilation facilitate an increase of flow and decrease the resistance by enlarging the airways' cross-sectional area. But friction, turbulence, in homogeneous distribution of the resistance along airways and alveoli, and airway cross-sectional area changes induced by compressive transmural pressures become also of increasing importance once ventilation increases. The importance of friction and turbulence have been demonstrated by experiments where exercising horses breathed a helium-oxygen mixture. Having a lesser density than air and therefore minimizing turbulence and friction, it induced a significant increase in minute ventilation [118,125], due to an increase in respiratory frequency, as well as a 50% decrease in the total pulmonary resistance and mechanical work of breathing .
At low exercise intensity, physiological and physical factors cancel each other and the resistance remains unchanged, while during heavy exercise, the physical factors largely override the physiological ones and the resistance increases.
Studies of the inspiratory and expiratory components of the resistance values in trotting horses have shown that, during inspiration, the extra-thoracic airways account for more than 90% of the total pulmonary resistance while, during expiration, the intra-thoracic airways are responsible for more than 50% of the pulmonary resistance (Fig. 13) . This observation could be explained by the fact that, during exercise, a dynamic partial collapse may occur when the pressure surrounding the airways exceeds the pressure within the lumen . When a horse inhales, sub-atmospheric pressures in the extra-thoracic airways may be as low as minus 5 kPa while the pressure in the surrounding tissues remains atmospheric. During expiration, the intra-thoracic pressure becomes greater than the pressure prevailing inside some of the intra-thoracic airways. When exposed to compressive pressures, these structures tend to collapse, consequently increasing their resistance to airflow.
If this collapse occurs normally in healthy horses, it can be expected to be dramatically worse in horses suffering from either upper or lower airway obstruction, a condition accompanied by substantial transmural pressures during exercise [20,124,128].
It has been shown that both the extra- and intra-thoracic parts of the trachea are sufficiently compliant to decrease their cross sectional areas when submitted to high, but nevertheless physiological, compressive transmural pressures [24,129]. Moreover, the shape of the cross sectional area of the individual trachea significantly influences collapsibility: Tracheae with a circular cross sectional shape are less compressible than tracheae with a more ellipsoidal shape . This is particularly important in view of the variability observed in this shape among individual horses, some horses being probably more susceptible than others to dynamic tracheal collapse.
Last, the extension of the trachea decreases its collapsibility . This means that hyperextension stiffens the trachea under dynamic conditions, therefore decreasing its resistance to airflow and consequently increasing the maximal airflow. This could explain the benefit of neck extension during maximal exercise: The consequent longitudinal extension of the trachea decreasing the tracheal compliance and minimizing the phenomenon of dynamic collapse.
The dynamic compliance gives an estimate of the elastic properties of the lung. The lung has an inherent elasticity due to the tissue elasticity (the normal lung is an elastic structure that contains a network of elastin and collagen fibres) and the surface tension forces. The latter is lowered by the pulmonary surfactant, a complex material composed of 80% lipids and 20% proteins.
Although dynamic compliance depends on the intrinsic properties of the lung, it is also influenced by dynamic factors like lung inflation and respiratory frequency. Dynamic compliance increases with lung inflation and may decrease with increasing respiratory frequency. The latter is especially true in lungs presenting a certain degree of sub-obstruction of the lower airways. Therefore, dynamic compliance measurement is sometimes used as an index of ventilatory asynchronism.
The dynamic lung compliance at rest is approximately equal to 23 L/kPa. Because of (1) technical problems associated with the determination of dynamic compliance during hyperventilation in large animals, and (2) the magnitude of the pressure necessary for flow acceleration, the measured compliance during exercise may, once a high level of ventilation is reached, become negative . Recently, Bayly and Slocombe , reported that the dynamic compliance of horses running at 10 m/sec, calculated taking into account the inertial pressure, seems to be 25% of its pre-exercise value. However, whether this decrease is the genuine reflection of changes in elastic properties of the horse's lung, or due to an increase in respiratory asynchronism, or related to other technical problems is not yet elucidated.
Inertial forces are those necessary to accelerate or decelerate the air in the respiratory airway. In human beings, the inertance of the respiratory system is negligible, and so are the pressures necessary to induce accelerations and decelerations of the air in the airways (inertial pressures), even during intense exercise. In contrast, the inertial pressures are not negligible in exercising horses  namely because of their tracheal design. Actually, the tracheal diameter should ideally satisfy dominant constraints such as tracheal resistance, inertance (the longer the trachea and the smaller its section, the greater its inertance), flow limitation, dead space and minimum work of breathing . A larger diameter would be advantageous with respect to pulmonary resistance and inertance but disadvantageous for the anatomic dead space.
During quiet breathing, respiratory frequency is about 14/min, tidal volume about 5.5 L, the total pressure change to which the lungs are subjected about 0.44 kPa and the pressure associated with volume acceleration about 0.02 kPa . During a fast gallop, respiratory frequency increases up to 121/min, tidal volume up to 13.2 L; the maximal pressure change reaches values of 8.5 kPa and total volume acceleration more than 3000 L/sec2. The pressure required to produce these accelerations is then 4.3 kPa or 50% of the total pressure change. Therefore, it appears that inertial pressures become of great importance in the exercising horse. They may even be limiting or at least a constraining factor to any further increase in ventilation .
A classical approach to the assessment of the mechanical work of breathing is based on the measurement of the area of the pressure-volume loops. Although it is well known that the total work of breathing is underestimated by this method, the latter gives a good estimation of the dynamic components of the work of breathing. It has been shown that the work per respiratory cycle, the work per litre of air ventilated and the work per minute are dramatically increased during exercise . For example, the power output (i.e., the work of breathing per minute) increases 475 times between rest and a strenuous gallop .
The relationship between the mechanical work of breathing per minute and the minute volume in running horses is curvilinear with an upwards concavity (Fig. 14).
Figure 14. Individual curves of the relationship between the work of breathing per minute (Wrm) and the expired minute volume (Ve) in eight ponies running at increasing speed .
The curve is of ever-increasing slope, implying that the mechanical cost of breathing for any additional units of air ventilated becomes greater with any increase in ventilation [35,133]. These observations strongly suggest that the work of breathing could be a limiting or at least a constraining factor to further increases in ventilation during strenuous exercise.
The sharp increase in the work of breathing with exercise-induced hyperventilation is explained by the increase in resistive, elastic and inertial work. The increase in the resistive work is related to the increase in the total pulmonary resistance. The 3-fold increase in tidal volume probably increases the elastic work significantly  due to chest wall stiffness. Furthermore, the length of the airways and the magnitude of flow accelerations during exercise make the inertial work important .
Last, the ratio of the mechanical work of breathing/O2 uptake has been calculated in galloping horses, in order to evaluate the relative respiratory muscle O2 uptake compared with total O2 uptake. This ratio increases exponentially with the minute volume indicating that during exercise, the respiratory muscles O2 uptake reaches a substantial percentage of the O2 uptake . This suggests that in horses as in man , there is a so called "critical level of ventilation" above which any further increase in O2 uptake would be entirely consumed by the respiratory muscles.
Control of Breathing
In healthy horses at rest, blood gases and chemical composition of the blood remain remarkably steady. The matching of the metabolic needs and the alveolar ventilation is under the control of a central controller, which receives afferents coming from peripheral and central receptors, regulates via motor neurons, the ventilatory muscles according to the received information. The respiratory control center is located in the pons and in the medulla, and its activity is modulated by a variety of neural inputs. Higher conscious centers can also modify the pattern of breathing.
Small changes in arterial partial pressure of CO2 and/or pH, detected by both peripheral and central chemoreceptors, are more potent regulators of ventilation than changes in arterial O2 partial pressure, detected by peripheral chemoreceptors only.
The peripheral chemoreceptors are located in the carotid bodies at the bifurcation of the common carotid arteries and in the aortic bodies, near the aortic arch. They send afferent impulses to the control center via the vagus and the glosso-pharyngeal nerves. Their activity is enhanced by hypercapnia, hypoxemia and acidosis, but also by hyperthermia and decreased blood pressure. While their response to CO2 and pH is quite linear, their response to changes in arterial O2 partial pressure is non-linear. They only show enhanced activity once arterial O2 partial pressure is below 60 mmHg.
The central chemoreceptor tissue is located near the ventral aspect of the medulla. It lies in the intracerebral interstitial fluid and is separated from the blood by the brain barrier. It apparently responds to changes in the interstitial tissue fluid pH. The latter is induced either by changes in arterial partial pressure of CO2, giving a fast response (because CO2 diffuses freely through the blood brain barrier) or by a change in blood pH, giving a delayed response (because the barrier is relatively impermeable to H+). Therefore an acute increase of H+ concentration is at first detected by the peripheral receptors.
The pulmonary stretch receptors are nerves ending in the tracheal and bronchial smooth muscles. Their activity is enhanced by the enlargement of the airway cross section, for example when the lung volume increases, and results in an inhibition of further inspiratory activity. The stretch receptors could be responsible for the adjustment of the pattern of breathing to minimize the energetic cost of breathing. They could also prevent a lung overstretching when the ventilatory demand is high, i.e., during heavy exercise.
The question of how the ventilatory rate is controlled during exercise is one of the major unresolved issues in respiratory physiology. The controversy mainly comes from the origin of the stimulus that provides for a rapid and precise adjustment of alveolar ventilation to meet the metabolic demand. In humans, there are several theories proposing either neural stimuli, or humoral stimuli, or a combination of both . In horses, the control seems to be different according to the exercise intensity.
During short term moderate exercise, arterial blood gases and chemical composition do not change. This stability results because the ventilation rate increases in parallel with the metabolic rate. However, gas tensions are far too stable to account for the increase in ventilation on the basis of the simple negative feed-back system existing at rest. Although there is a considerable conviction that gas tensions (especially partial pressure of CO2 and associated H+ concentration) are involved in the control of ventilation during exercise as well, how they are involved remains enigmatic. It is likely that other drives such as mechano-reflexes originating from motion of the working limbs, changes in cardiac output, thermoregulation, and cortical and psychological factors are involved in the control of ventilation .
The exercise-induced hyperpnea at mild and moderate exercise intensities has been thoroughly studied in ponies, showing that in this species, the hyperpnea is related to an increase in lactic acidosis  and to spinal afferent information . Exercise-induced hyperpnea is not related to increases in arterial or venous CO2 [139-143], a decrease in blood pH , an increase of H+ stimulation at the medullary receptors , a decrease in arterial O2 partial pressure , a cardio-vascular cause  nor an influence of limb motion . However, transposition of these observations to horses is questionable because horses and ponies differ in their respiratory adjustments. While the horse becomes hypoxemic and sometimes hypercapnic during heavy exercise, the pony does not become hypoxemic and on the contrary becomes hypocapnic, with arterial partial pressures of CO2 as low as 27 mmHg [117,148].
During strenuous exercise, the horse, independent of its ability and state of fitness, demonstrates a decrease in arterial O2 partial pressure and pH and sometimes an increase in arterial partial pressure of CO2. These chemical regulators are supposed to strongly stimulate the ventilation, but the stimuli appear to be insufficient to maintain arterial blood gas homeostasis. This is not the case in other species like man, dog and pony which do not show hypoxemia or hypercapnia during strenuous exercise. However, it must be pointed out that in the elite, endurance, human athlete at peak fitness, cardio-vascular and muscular adaptations to training reach such an exceptional level that the pulmonary system may be taxed maximally or even lag behind the functional capacity of the remaining aerobic system. Such a condition may result in hypoxemia and hypercapnia during high intensity exercise .
Thus, in contrast to other species, the horse does not adopt a compensatory hyperventilation, i.e., a strategy which could compensate for the gas exchange impairment [88,116]. Working in hot and humid conditions still exacerbate this hypoventilation  (Fig. 15). The reason for the hypercapnic hypoventilation in the horse remains unclear and numerous hypotheses are put forward.
Figure 15. Expired minute volume (VE), tidal volume (VT) and respiratory frequency (f) measured in 5 Standardbred horses during a standardized treadmill test in temperate conditions (TC) and in hot and humid conditions (HHC) .
The locomotion-respiration coupling has been suggested as a major constraint to the increase in ventilation. Although there is no doubt that respiratory frequency is totally related to step frequency in galloping horses, several experimental observations rule out the coupling as a reason for hypoventilation: (1) Standardbred horses, racing at a trot (a gait where a coupling may exist but is not compulsory), also demonstrate hypoxemia and hypercapnia [41,86,151]; (2) the magnitude of hypercapnia is poorly related to the respiratory frequency ; (3) the respiratory frequency of the pony is also tightly coupled with its step frequency and, nevertheless, it adopts a compensatory hyperventilation and becomes hypocapnic during heavy exercise .
A slight increase in ventilation in horses running at 10m/sec is observed when the CO2 concentration in the inhaled air increases from 0 to 3%. But a further increase in inhaled CO2 up to 6% does not induce any further changes in ventilation, when these horses were not at their maximal ventilation capacity , suggesting a lesser sensitivity of the chemoreceptors. However, the underlying mechanisms of this observation remain to be elucidated.
Theoretically, the compensatory hyperventilation which would be required for the homeostasis of arterial O2 partial pressure should be ensured by either (1) a tidal volume of about 23 L, which together with a respiratory frequency of about 120 breaths/min implies mean respiratory airflow of more than 100 L/sec, or (2) a coupling 2:1 between respiratory frequency and step frequency, which implies that the respiratory frequency should reach values of about 240 breaths/min . In terms of respiratory muscle energetics and force-velocity characteristics, such a level of ventilation would probably be impossible to reach.
During high level ventilation, the energy demand of the respiratory muscles becomes substantial and the energy supply may be insufficient to satisfy this demand. This could result in a negative metabolic balance at the level of the ventilatory muscles, which could lead to fatigue . This phenomenon could control the pattern of breathing by a negative feed-back mechanism acting either on the respiratory centers or directly on the ventilatory muscles . This negative feed-back would override all the other positive feed-back, tending to further increase the ventilation .
The occurrence of muscle exhaustion has already been demonstrated to occur in man after high intensity short term exercise  as well as during prolonged exercise . This results in a progressive increase in diaphragmatic excitation-contraction decoupling [155,157]. Although it has been shown that, in the horse during high intensity short-term exercise, occurrence of exhaustion is associated with a sudden decrease in the minute volume and the O2 uptake [60,158], the occurrence of muscle fatigue remains to be demonstrated in equids.
To summarise, the fact that the increase in ventilation becomes limited during heavy exercise in horses may be explained by several factors. First, it has been shown that, during strenuous exercise, the horse reaches a "critical level of ventilation" above which any further increase in O2 uptake would be entirely consumed by the respiratory muscles . The advantage of not increasing ventilation further during strenuous exercise may be that extra-flow resistive and elastic work is avoided and that there is a reduction in the O2 uptake of ventilatory muscles, consequently reducing their energy demand. In terms of performance, hypoxemia and hypercapnia could be less disadvantageous than reaching the critical level of ventilation. Second, this negative feed-back mechanism could protect the ventilatory muscles against exhaustion and irreversible damage to the contractile oxidative machinery. Third, pulmonary overstretching, i.e., high lung volume, has been shown to increase the fragility of alveolar and vessel walls . By limiting the increase in lung volume, the horse could minimize the risk of tissue rupture possibly leading to severe exercise-induced pulmonary hemorrhage.
During prolonged heavy - but not maximal - exercise, the impairment of gas exchange (especially hypercapnia) seems to be progressively compensated after several minutes of exercise [116,160].
This suggests that the horse, like the human, experiences a "second wind" phenomenon, i.e., the relief of hypoventilation which could be induced by diaphragmatic fatigue . Several factors are put forward to explain the improvement in ventilatory muscle function after a few minutes of exercise: (1) the length of the diaphragm may be modified by recruitment of other ventilatory muscles or by changes in the functional residual capacity, resulting in an increase in its force of contraction according to its length-tension characteristics; (2) the contractile function of the diaphragm may be improved; (3) the blood flow in the working diaphragm may be redistributed and (4) the catecholamine release may lead to increased contractility. These assumptions remain to be confirmed in the horse.
3. Respiration During Exercise Recovery
After exertion, all physiological measurements return progressively to their resting values, the speed of this return being dependent on both intensity and duration of the exercise performed, the state of fitness of the horse and the bioclimatologic conditions.
The metabolic reasons for the "excess post-exercise O2 uptake" are resynthesis of phosphocreatine in the muscles, catabolism or anabolism of blood lactate, persistence of high body temperature and restoration of hormonal reserves . This excess O2 uptake is associated with an elevated minute volume which is mainly the result of an increase in respiratory frequency . Obviously, 5 min after exercise, horses hyperventilate, as assessed by the high ventilatory equivalent for O2. Despite a high dead space to tidal volume ratio, this hyperventilation results in alveolar hyperventilation and a consequent hyperoxia and hypocapnia [35,86]. This hyperventilation and the resulting respiratory alkalosis could be advantageous to compensate for the metabolic acidosis.
The thermoregulatory role of the respiratory system also accounts for the post-effort tachypneic hyperventilation. It has been shown that ponies recovering from the same treadmill exercise test in hot and humid conditions have a significantly higher respiratory frequency than ponies recovering in dry and cold conditions . This must be taken into account when horses running endurance courses are examined at the "vet-gate": When bioclimatological conditions are hot and humid, the respiratory frequency is a poor indication of the actual ventilatory demand of the horse [151,164].
4. Respiratory Adaptations to Training
Training rapidly and significantly improves maximal oxygen consumption [36,90,165,166]. The cardio-vascular mechanisms underlying the improvement in O2 uptake with training have been already studied, but the accompanying ventilatory changes have been less investigated. However, some information is now available.
Training does not improve the exercise-induced alterations in blood gas tensions during and after heavy exercise [86,90,167] and on the contrary, hypoxemia and hypercapnia are more pronounced after a training period [158,168]. The pulmonary arterial pressure and the pulmonary blood flow velocity during mild standardised exercise are also unchanged .
The significant increase in the O2 uptake induced by training is not accompanied by an equivalent increase in the minute volume. In Thoroughbred horses, the ventilatory rate is unchanged during a maximal exercise test up to fatigue after a period of training (Fig. 16) [36,90,168].
Figure 16. Peak oxygen consumption (VO2) and expired minute volume (VE) in 10 Thoroughbred horses before (UnT) and after a 15 week-training program (T) showing a significant increase of the first parameter while the second remain unchanged .
This results in a significant decrease in the ventilatory equivalent for O2 (minute volume to O2 uptake ratio). This is obvious even in the early stages of training . The mechanisms underlying this training adjustment are still obscure. These results suggest that pulmonary function could be a limiting factor to performance in the well trained horse (Fig. 17).
Figure 17. Training improves all the physiological steps of the oxygen chain except ventilation. Therefore, in well trained horses, the respiratory system is most probably the limiting factor on performance.
In human athletes, the improvement in the ventilatory equivalent for O2 is explained by a training-induced reduction in respiratory frequency and increase in tidal volume. Therefore, the time for gas exchange at the level of the alveoli is increased by training, and this induces a better alveolar O2 extraction- the mean expired O2 is about 18% in untrained men and 14% in well trained athletes .
In equine athletes, the decrease in the ventilatory equivalent after training is also associated with an improvement in the alveolar O2 extraction . However, in contrast with humans, the reason for this improvement seems not to be a change in the pattern of breathing but more probably an increase in the affinity of hemoglobin for O2. This could be explained by a training-induced shift to the left of the oxyhemoglobin curve resulting from a decrease in the severity of the exercise-induced acidosis and hyperthermia .
Whatever the reason, the decrease in the ventilatory equivalent means that horses breathe less air to ensure a given O2 uptake. Theoretically, this could imply that (1) the relative energetic cost of ventilation is reduced and/or (2) the fatigue of the respiratory muscles is delayed and/or (3) the reduction in O2 uptake by the muscles is profitable to the working locomotor muscles. However, these assumptions remain to be proved.
After a 3-week period of detraining, most of the training-modified ventilatory parameters, i.e., O2 uptake, ventilatory equivalent for O2 and minute ventilation during a standardised exercise, return to their pre-training level [36,166]. This indicates that, while the ventilatory adaptations occur rapidly when the intensity of training is sufficient, they are nevertheless transient and highly reversible.
5. The Respiratory System and its Thermoregulatory Role
Many mammals use the respiratory system to lose heat by evaporative cooling. To increase heat loss, they expire through the mouth instead of through the nose and they hyperventilate by increasing the respiratory frequency and reducing tidal volume. Horses cannot breathe through the mouth but some anatomic and physiologic characteristics provide evidence that the respiratory system plays a role in body thermoregulation at rest, during exercise and recovery.
In resting ponies, changes in ambient temperature induce modification in respiratory frequency and tidal volume, even when body temperature remains constant. Changes in skin and airway temperatures therefore appear capable of eliciting changes in breathing .
Prolonged steady exercise induces a progressive increase in respiratory frequency with an increase in the physiological dead space to tidal volume ratio [59-61,172]. This suggests that, in horses as in man , the respiratory system becomes increasingly involved in thermoregulation during long term effort. Other experiments confirm the thermoregulatory role of the respiratory system in the horse during exercise. Standardbred horses exercised in extremely cold conditions (minus 25ºC) reduce their respiratory frequency during the early stages of exercise and recovery . Ponies performing the same test in hot and humid conditions recover with a higher respiratory frequency than in dry and cold conditions . The same has been observed in Standardbreds recovering from an exercise in hot and humid environmental conditions .
Hypothalamic temperature increases more in exercising horses when upper respiratory tract is bypassed, suggesting a selective brain cooling via blood cooled within the upper airway  including probably the guttural pouches [10,173].
During prolonged exercise in ponies, the bronchial circulation has been shown to increase progressively as core temperature increased with exercise duration [176,177]. Indeed, the bronchial arteries form a circulatory plexus in the connective tissues along the airways, the role of which is to ensure some heat dissipation. The same study shows a lesser modification in tracheal circulation, suggesting the heat exchange occurs mainly at the level of the bronchi. Last, it has been shown that the heat loss from the pulmonary circulation increases as exercise progresses at all intensities .
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Affiliation of the authors at the time of publication
1,3Department of Physiology and Sport Medicine, Faculty of Veterinary Medecine, Liège, Belgium
2Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Washington State University, Pullman, WA, USA.