Heart Rate, Hematological, and Biochemical Responses to Exercise on Water Treadmill with Artificial River in School Horses

Abstract

Water treadmill (WT) exercise is gaining popularity among equine athletes as it allows for increased workload through the resistance posed by water. However, the effect of an artificial river (AR), which further increases this resistance, on equine fitness indicators has not yet been investigated. This study aimed to determine whether WT exercise with varying water depths and the presence of an AR influences physiological response indicators. Fifteen school horses (n = 15) underwent five treadmill exercise sessions: on a dry treadmill (DT), in fetlock-depth water with and without AR, and in carpal-depth water with and without AR. Physiological responses were assessed pre-exercise, during the highest workload, and at 30 min and 24 h post-exercise by measuring heart rate (HR), blood lactate concentration (LAC), red blood cell count (RBC), hemoglobin concentration (HGB), and the activity of three serum enzymes: creatine phosphokinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). HR and LAC increased significantly (p < 0.0001) from the pre-exercise resting state to the highest workload time point during treadmill exercise, regardless of session type. During the carpal-depth WT+AR session, horses achieved a fitness level characterized by the highest HR (p < 0.0001), LAC (p < 0.0001), and LDH activity (p = 0.001). Thus, horses’ physiological responses to walking on a WT with AR differ from those observed on a DT and a WT without AR. WT exercise with AR represents a low-to-moderate intensity aerobic workload for horses, which increases with water depth from fetlock to carpal levels. However, further research is required to evaluate its inclusion in training or rehabilitation programs for athletic horses and its potential beneficial effects.

1. Introduction

Water treadmill (WT) exercise is becoming increasingly popular in the conditioning [1,2,3,4] and rehabilitation [5,6,7,8] of equine athletes. The decision to incorporate WT exercise into a conditioning or rehabilitation program is often made to leverage the benefits of water properties [9]. When a horse is immersed in water, buoyancy acts against the horse’s body weight [10] and reduces vertical acceleration and vertical ground reaction forces [11]. Additionally, the hydrostatic pressure can enhance the horse’s ability to maintain postural control [5]. Finally, the drag force opposing the horse’s forward movement modifies the kinematics of a horse’s limbs [4,12,13,14,15,16] and back [4,16,17,18,19] and increases energy expenditure during exercise [2,20,21,22,23].

WT exercise protocols vary based on the combination of water depth, belt speed (and consequently the horse’s gait), exercise duration, and frequency of application, producing different effects on the horse working in water. Therefore, like any other aspect of a horse’s exercise regimen, WT exercise protocols should be tailored to address specific individual exercise objectives [4]. If the goal of a WT exercise is to encourage the horse to take fewer, longer strides with increased swing duration and enhanced lumbar flexion, walking in fetlock-depth water is recommended [18,24]. To reduce limb protraction, walking in carpal-depth water is advised [4,16]. To increase limb flexion and back vertical displacement, walking in stifle-depth water is preferable [4,15,17]. It can be observed that water depth is the primary factor influencing WT exercise outcomes. However, increased belt speed promotes greater craniocaudal movement of the head and neck, potentially negating improvements in limb and back kinematics, particularly at higher water depths [4]. Excessive craniocaudal movements of the head and neck indicate that belt speed is too high, at any water depth [25]. Therefore, from a kinematic perspective, high belt speed exercises in water should be avoided [4].

On the other hand, to progressively develop a horse’s cardiovascular and muscular fitness, it is recommended to gradually increase energy expenditure [26]. Energy expenditure during the work on the WT was previously investigated using indicators of physiological responses, such as heart rate (HR) [2,12,20,21,22,23,26,27,28,29] as well as hemoglobin concentration (HGB) [20], lactate concentrations (LAC) [2,20,21,22,23,26,30], creatine phosphokinase (CK) activity [28,30], aspartate aminotransferase (AST) [30], and lactate dehydrogenase (LDH) [30] in blood. However, it is important to highlight that the primary purpose of these hematological and biochemical indicators is to differentiate pathological alterations from physiological ones. While their variations within normal reference ranges may aid in assessing physiological responses to effort on the WT [2,12,20,21,22,23,26,27,28,29,30], they do not necessarily indicate an improvement in the horse’s health or wellbeing. These studies on energy expenditure showed that the WT exercise represents a low- to moderate-intensity aerobic activity [2,20,21,22,23,26,27,28], and the greatest workload can be achieved by increasing the resistance posed by the water. To date, this increased resistance has been achieved by increasing water depth [2,4,23,26] or by increasing the treadmill belt speed [22,23,26], not by introducing the water counterflow of an artificial river (AR).

One may observe that even a single WT exercise session increases muscle activity, indicating greater muscular workload against the water’s resistance [31]; however, exercise intensity changes with the combination of WT settings. Walking in fetlock-depth water is initially associated with the horse adapting to this form of locomotion by attempting to avoid contact with the water, effectively ’stepping over the water surface’ [13,15,18]. This locomotion strategy enables horses to move forward with minimal energy expenditure when only partially immersed in water [9]. Walking in carpal-depth and stifle-depth water increases oxygen uptake [23] and can be incorporated into conditioning programs [2]. Trotting in stifle-depth water significantly increases energy expenditure [22,26]; however, from a kinematic perspective, it is contraindicated [4,25]. However, both physiological and kinematic responses have not yet been investigated while walking and trotting in fetlock-, carpal-, and stifle-depth water on the WT with active water counterflow, which the horse must counteract. We hypothesize that incorporating an AR into the WT exercise increases the horse’s energy expenditure.

Given the lack of data on equine workload during WT exercise with an AR, this study aims to determine whether WT exercise with a walk belt speed, varying water depths, and an AR affects physiological response indicators in school horses.

2. Materials and Methods

2.1. Horses

The study involved 15 healthy school horses from the riding school of the Didactic Stable of Horse Breeding Division at the Warsaw University of Life Sciences (WULS). The horses ranged in age from 6 years to 18 years (median age: 12 years) and had a wither height between 152 cm and 168 cm (median height: 166 cm). Among the group, eight horses (53%) were geldings, and seven horses (47%) were mares. All horses were Warmblood breeds, with ten horses (67%) being of the Polish half-bred and five horses (33%) being of the Malopolska breed. The sample size was calculated using the formula for quantitative data [32] and based on data from a previous study [25], which examined the difference in HR measured on a treadmill under dry conditions (mean: 41.2; range: 28.4–54.1 beats per minute, bpm) and with water at carpal depth (mean: 51.7; range: 46.1–60.5 bpm) during exercise at a belt speed of 1.25 m/s. These calculations, assuming a 95% significance level (p ≤ 0.05) and 80% study power, indicated a required sample size of 14.53. Consequently, 15 horses were enrolled in this study, aligning with the reference study [25], which also conducted repeated measures on 15 horses.

Horses were recruited based on the results of physical and orthopedic examinations. The basic physical examination followed international veterinary standards [33] and included assessment of rectal temperature, heart rate, respiratory rate, mucous membranes, capillary refill time, and lymph nodes. The lameness was assigned according to the American Association of Equine Practitioners (AAEP) lameness scale [34]. Exclusion criteria included physical parameters outside the physiological range [33] and a lameness score of 1/5 or greater [34]. None of the horses met the exclusion criteria. All had no clinical history of recent lameness or poor performance.

All horses were owned by WULS and housed under identical conditions and management in the Didactic Stable of the Horse Breeding Division at WULS. The horses were fed individually calculated rations of hay, oats, and concentrate based on their nutritional requirements, with feed distributed across three meals per day. A mineral salt block and fresh water were available at all times. During the WT study period, all horses were engaged in a daily leisure workload at the riding school of the Didactic Stable, including one hour of leisure riding per day, five days a week. In addition to under-saddle work, the horses had daily access to a sandy paddock for a minimum of six hours per day.

2.2. Water Treadmill Exercise

Horses were exercised on a WT (Technohorse Sp. z o.o., Skarżysko–Kamienna, Poland) equipped with an AR system that created a water counterflow using four water jets. These jets forced water to flow at a speed of 3 m/s opposite to the horse’s movement (AR mode). When the AR mode was activated, the water continuously flowed at a speed of 3 m/s. WT exercises were conducted following the guidelines for WT exercise in healthy horses [24].

At the start of this WT study, all horses were inexperienced with WT exercise. According to Nankervis et al. [27], horses were habituated to the WT exercise protocol before data collection to ensure accurate measurements of physiological responses. Horses were considered habituated once they exhibited no signs of stress and demonstrated regular movement. The habituation protocol, adapted from Greco–Otto et al. [23], included three initial WT exercise sessions of at least 20 min each. During the first session, horses were walked on the treadmill with a dry belt. In the second session, horses were walked in fetlock-depth water, with AR mode activated during the last 5 min of exercise. In the third session, horses were walked in carpal-depth water, with AR mode activated during the last 5 min of exercise. After completing these three sessions, all horses were deemed habituated.

After completing the habituation process, each horse participated in five WT exercise sessions, conducted once per week. Each training session lasted 20 min, with a belt speed of 1.25 m/s, excluded filling and emptying time (5–10 min depending on water depth). Water depth was determined using anatomical landmarks and adjusted to each horse individually. The first exercise session was performed on a dry treadmill (DT session) and served as a control. The second and third WT sessions were conducted at fetlock-depth water, with the AR mode activated during the third session. These were designated as the fetlock-depth WT session and the fetlock-depth WT+AR session, respectively. The fourth and fifth WT sessions were conducted at carpal-depth water, with the AR mode activated during the fifth session. These were designated as the carpal-depth WT session and the carpal-depth WT+AR session, respectively. Throughout the WT study period, all horses continued their normal workload and had free access to the paddock for movement.

2.3. Assessment of the Physiological Responses

2.3.1. Heart Rate

HR (bpm) was continuously monitored during treadmill exercise using a Polar Equine system (Polar Electro Oy, Kempele, Finland). The electrode was placed on the left side of the horse’s thorax, just behind the front limb and the HR sensor was positioned on the elastic belt fastened around the thorax just behind the withers. The mean HR was calculated over a 60 s measurement at four time points: before the WT session [35], during the final 60 s of each treadmill session [23], and at 30 min and 24 h post-treadmill session.

2.3.2. Hematological and Biochemical Responses

Blood samples were collected via jugular venipuncture using a BD Vacutainer^® system (Plymouth, UK). The blood samples were collected in the following order: K2-EDTA tubes first (BD Vacutainer^®, Plymouth, UK) for hematological analysis, followed by dry tubes (BD Vacutainer^®, Plymouth, UK) for biochemical analysis. A total of 5 mL of blood was collected per tube. Blood samples were collected once at each time point: before the treadmill session, immediately after the treadmill session, and at 30 min and 24 h post-treadmill session.

Samples in EDTA and dry tubes were stored at +4 °C and sent to a reference laboratory (Vetlab sp. z o.o., Warsaw, Poland) within no more than 2 h of blood collection for hematological and biochemical analyses, respectively. From the hematological analysis, red blood cell count (RBC, ×10¹²/L) and HGB (g/dL) were measured using an ABC Vet analyzer (Horiba Medical, Minami-ku, Japan). For the biochemical analysis, CK (U/L), AST (U/L), and LDH (U/L) activities were evaluated using a Miura One analyzer (ISE. S.r.l., Albuccione, Italy). The normal reference ranges were considered as follows: RBC, 2 to 12 × 10¹²/L; HGB, 8 to 17 g/dL; CK, 90 to 565 U/L; AST, 1 to 450 U/L; and LDH, 0.1 to 639 U/L. Lactate concentration (LAC, mmol/L) was analyzed in whole blood immediately after sample collection using an Accusport^® device (Roche Diagnostics, Basel, Switzerland). If the device returned a LAC value below the detectable level, annotated as low, the value 0.5 mmol/L was assigned.

2.4. Statistical Analysis

Statistical analysis was performed using Prism software version 6.01 (GraphPad Software Inc., San Diego, CA, USA). The distribution of each data series was tested using the Shapiro–Wilk normality test. As some data series did not follow a normal distribution, data are presented in tables using medians and ranges (minimum and maximum values).

The effects of treadmill exercise and recovery were tested independently for each treadmill exercise session by comparing physiological response measures across time points. The effects of treadmill settings were tested across treadmill exercise sessions by comparing physiological response measures at the same time points between treadmill exercise sessions. In both cases, data series were analyzed as paired data using either a one-way repeated measures ANOVA summary or a Friedman test, depending on the data distribution. The repeated measures ANOVA summary was applied when a normal distribution was confirmed for all compared data series. When at least one data series did not follow a normal distribution, the Friedman test was used. If significant differences were found, post hoc tests were performed. Tukey’s multiple comparisons test followed the repeated measures ANOVA summary, while Dunn’s multiple comparisons test followed the Friedman test. Statistical significance was set at p < 0.05.

3. Results

3.1. Exercise Time Points Effect

HR increased during treadmill exercise, rising from the resting state before the session to the final 60 s of each session, regardless of the session type. Following exercise, HR gradually decreased and was lower at 30 min post-exercise compared to the final 60 s of each session for both fetlock-depth WT sessions and the carpal-depth WT+AR session. For the DT and carpal-depth WT sessions, no significant differences were observed between these two time points. For all sessions, HR measured 24 h post-exercise was lower than during exercise and at 30 min post-exercise. Furthermore, HR at 24 h post-exercise did not differ from the resting HR recorded before exercise. Similarly, LAC increased during treadmill exercise, rising from the resting state before the session to the sampling point immediately after the session, regardless of the session type. For most sessions, LAC decreased by 30 min post-exercise, except for the carpal-depth WT+AR session. In this session, no significant differences in LAC were observed between the immediate post-exercise and 30 min post-exercise sampling points, or between the 30 min and 24 h post-exercise sampling points. Across all sessions, no significant differences in LAC were observed between the pre-exercise and 24 h post-exercise sampling points (Table 1).

RBC showed variations across time points, with higher values observed before and immediately after exercise compared to 30 min and 24 h post-exercise for both fetlock-depth WT sessions. Additionally, RBC was higher immediately after exercise than at other time points for the carpal-depth WT+AR session. No significant differences in RBC were noted across time points for the remaining sessions. HGB also varied across time points. Higher values were observed immediately after exercise compared to all other time points for the DT session. For the fetlock-depth WT session, HGB was higher before and immediately after exercise compared to the remaining post-exercise time points. Similarly, for the fetlock-depth WT+AR session, HGB values were higher immediately after exercise compared to the remaining post-exercise time points. No significant differences in HGB were observed across time points for the other sessions (Table 2).

CK activity was higher 24 h post-exercise compared to pre-exercise for the DT session, as well as immediately post-exercise compared to pre-exercise for all WT sessions. For both DT and WT sessions, no significant differences in CK activity were observed among the post-exercise time points. AST activity was higher immediately and 30 min post-exercise compared to pre-exercise for the DT session. For the fetlock-depth WT session and the carpal-depth WT+AR session, AST activity was higher at all post-exercise time points compared to pre-exercise. LDH activity was higher at 30 min and 24 h post-exercise compared to pre-exercise, with no significant differences observed among the post-exercise time points for the fetlock-depth WT session. No other significant differences in the measured enzyme activities were observed across time points (Table 3).

3.2. Treadmill Settings Effect

No significant differences were found at the pre-exercise time point between exercise sessions for any of the measured indicators of physiological response (Table 1, Table 2 and Table 3).

At the final 60 s of each WT session, HR was higher compared to the DT session. Additionally, at this time point, horses exhibited higher HR during the carpal-depth WT+AR session than during both fetlock-depth WT sessions; however, no significant difference was observed between the carpal-depth WT sessions regardless of whether the AR mode was activated. Similarly, immediately post-exercise, LAC was higher for almost all WT sessions compared to the DT session, except for the fetlock-depth WT session, where no significant differences were found between the DT session and the fetlock-depth WT+AR session. No differences in LAC were observed at this time point between the fetlock-depth WT+AR session, carpal-depth WT session, and carpal-depth WT+AR session. At the 30 min post-exercise time point, HR was higher for the DT session than for both fetlock-depth WT sessions, with no differences observed between both carpal-depth WT sessions and the remaining sessions. At the same time point, LAC was higher for the carpal-depth WT+AR session compared to the DT session and the fetlock-depth WT session, with no significant differences among the remaining sessions. No significant differences in HR or LAC were observed 24 h post-exercise (Table 1).

Immediately and at 30 min post-exercise, no session-related differences in RBC or HGB were observed. However, differences in both RBC and HGB were noted at the 24 h post-exercise time point (Table 2).

Immediately post-exercise, CK activity was higher for almost all WT sessions compared to the DT session, except for the carpal-depth WT session, where no differences were found with the remaining sessions. At the same time point, no session-related differences in AST activity were observed. However, differences in LDH activity were noted at this time point. LDH activity differed between the DT session and both sessions with activated AR mode, as well as among sessions with activated AR mode. Additionally, both the fetlock-depth and carpal-depth WT sessions differed depending on whether the AR mode was activated. At the 30 min post-exercise time point, CK activity and AST activity showed no session-related differences, while differences in LDH activity were again observed. LDH activity differed between the fetlock-depth WT session and the fetlock-depth WT+AR session, as well as between the fetlock-depth WT+AR session and the carpal-depth WT+AR session. Similar enzyme activity was noted 24 h post-exercise (Table 3).

4. Discussion

It should be highlighted that, during the measurement of the highest workload, horses walking on a WT at fetlock-depth with an AR mode and at carpal-depth with and without AR achieved higher HR, LAC, and CK values than horses following a similar protocol on a DT. During each training session, HR and LAC reached levels previously reported for WT exercise at greater water depths [2,20,21,22,23,26,30], while CK activity remained within the normal range for all horses [28]. These findings support our hypothesis that incorporating AR into WT exercise increases the horse’s energy expenditure and workload. This observation aligns with previous research showing that the high degree of resistance created by water increases workload without adding concussive forces [2,36]. However, to date, a high degree of water resistance has primarily been achieved by increasing water depth [2,4,23,26] or by increasing the treadmill belt speed [22,23,26]. The current study demonstrates that increasing water resistance can also be achieved by introducing a water counterflow. This study highlights the potential for increased resistance created by water to influence horses’ physiological responses during water walking, encouraging further comprehensive research on the application of AR in WT exercise.

The findings of this study demonstrate the differential effects of the AR mode on WT exercise at two water depths in a group of healthy school horses. Fetlock-depth and carpal-depth were investigated based on previous studies indicating that water depths below the carpus are more commonly utilized than those above the carpus [1].

At fetlock-depth water, the only notable difference associated with AR mode activation was lower LDH activity. This may be related to the horses’ movement strategy on the WT at this depth. While walking in fetlock-depth water, horses tend to ‘step over’ the water surface to minimize contact with the water [13,15,18]. With AR mode enabled, this avoidance strategy may become even more pronounced, mitigating the assumed workload effect associated with increased water resistance. It can be speculated that avoiding water contact, regardless of flow rate, may remain effective at a constant belt speed. However, further kinematic studies [4,12,13,14,15,16,17,18,19,25] are necessary to confirm this hypothesis.

At carpal-depth water, more pronounced AR-related differences in physiological responses were detected. During the measurement of the highest workload, HR was higher during the carpal-depth WT+AR session compared to the DT and both fetlock-depth sessions. For the carpal-depth WT session without AR, no differences in HR were observed compared to the fetlock-depth sessions. LAC was elevated during both carpal-depth WT exercises compared to other treadmill exercises. Notably, in the carpal-depth WT+AR session, this increased LAC persisted for 30 min post-exercise, whereas it returned to resting levels at the same time point in the carpal-depth WT session and all other treadmill exercises. LDH activity was also higher immediately post-exercise in the carpal-depth WT+AR session compared to the session without AR.

However, the amount of LAC generated in any of the horses after WT exercises in this study does not meet the criteria for lactic acidosis. This suggests that horses were able to compensate for the increased energy expenditure and likely higher oxygen demand [2,23]. Previous research indicates that increased respiratory rate and enhanced oxygen transport capacity through the mobilization of the splenic RBC reservoir [20] act as compensatory mechanisms against increased blood LAC concentration in horses. However, in this study neither respiratory rate nor splenic contraction were assessed after effort to support this speculation. Although oxygen demand was not specifically measured in this preliminary study, oxygen consumption (V̇O₂) has been investigated in recent WT studies [2,23]. These studies demonstrated that walking at stifle-depth water increases HR and V̇O₂ compared to DT exercise and fetlock-depth WT exercise [23], suggesting potential conditioning benefits for Thoroughbreds [2]. Given the relatively low-intensity nature of WT exercises [2,20,21,22,23,26,27,28], such potential conditioning benefits requires walking [2,23] or even trotting [22,26] at higher water depths. However, higher depths may pose kinematic challenges and thus be contraindicated [4,25]. We speculate that horses undergoing carpal-depth WT exercise with AR could achieve similar submaximal physiological responses and cardiorespiratory fitness as those engaged in stifle-depth WT exercise without AR [2,23]. Therefore, further research utilizing ergospirometry systems [2,23,37] or impulse oscillation systems [37,38] is recommended to verify this hypothesis.

The speculation of splenic RBC reservoir mobilization [20] may be supported by the increased RBC levels observed immediately post-exercise in horses walking in the carpal-depth WT +AR session. A similar increase in RBC and HGB during walking at the DT and fetlock-depth WT, along with elevated HR 30 min post-exercise during the DT session, may also be attributed to a stress response. Similar to the Greco–Otto et al. [2] study, the variation in HR recovery times may be a result of differing stress levels. This is likely because the DT exercise was the first measured session following the completion of WT habituation. Previous studies have indicated that two habituation sessions to the WT exercise protocol are sufficient to ensure accurate physiological measurements [27]. In this study, however, three habituation sessions were conducted, following the protocol described by Greco–Otto et al. [23]. To further investigate the effects of WT exercise with AR, future studies should consider including cortisol measurements in blood or saliva as quantifiable indicators of stress in horses [39].

The absence of significant differences between exercise sessions for any of the measured physiological response indicators at the pre-exercise time point suggests that this study was well-designed, aligning with methodologies from previous publications [2,23,24,27,35,40]. However, awareness of the strengths of prior research highlights the limitations of this preliminary study. Physiological responses were measured in a group of warmblood school horses, which may limit the generalizability of the findings to other breeds or disciplines, such as show jumping [3,4,30], dressage [3,4], eventing [4], or barrel racing [23], as investigated in earlier studies. Extrapolation may also be challenging for Thoroughbreds [2,16] or Arabian horses [28], given that the physiological demands of school horses only partially align with those of endurance or racehorses [40]. Moreover, the block-assigned treadmill sessions, progressing from the least demanding DT exercise to the more demanding WT exercises with increasing water resistance, do not meet the criteria for a randomized controlled trial [41]. The decision to block the training sessions was made to limit the impact of stress on the horses [23,27], which could influence the results due to the progressively increasing workload. However, a certain effect resulting from this blocking approach is to be expected. To minimize this effect, WT exercise sessions were conducted once per week, allowing sufficient time for hematological and biochemical indicators to return to baseline levels. For this reason, the study should be considered preliminary, ranking lower on the Evidence-Based Medicine Ratings scale [41]. Future studies should aim to adopt a randomized model, if feasible, to strengthen the level of evidence.

Despite these limitations, the current findings provide insights that may aid in designing WT+AR exercise protocols for horses engaged in full sport or racing activities. These results could serve as preliminary guidelines for incorporating AR mode into equine training or rehabilitation protocols. However, because physiological responses were measured exclusively in healthy horses, the applicability of these findings to horses with pathological conditions remains uncertain. Further research is needed to evaluate the suitability of WT+AR exercise for conditioning and rehabilitation, particularly through more extensive research protocols. This would address the study’s primary limitations, including the lack of kinematic data [4,12,13,14,15,16,17,18,19,25] and ergospirometric measurements [2,23]. While this study demonstrated that incorporating AR into WT exercise increases workload intensity, it cannot yet be recommended for specific training or rehabilitation applications until further data on kinematic responses, muscle activity, and cardiorespiratory responses are available. Future research should investigate whether AR increases step length, limb protraction and retraction, joint flexion, and dorsoventral displacement of the back [4,12,13,14,15,16,17,18,19,25]. It is also essential to explore its impact on V̇O₂, tidal volume, minute ventilation, and breathing frequency [2,23] during walking at different belt speeds and water depths, such as carpal-depth and stifle-depth.

5. Conclusions

Horses’ physiological responses to walking on a WT with AR differed from those observed on a DT and a WT without an AR. WT exercise with AR represents a low-to-moderate intensity aerobic workload for horses, which increases as water depth rises from fetlock to carpal levels. During the carpal-depth WT+AR session, horses achieved a fitness level characterized by higher HR, LAC, and LDH activity, along with changes in RBC levels. WT exercise with AR does not result in metabolic overload for horses. However, its inclusion in training or rehabilitation programs for athletic horses and its potential beneficial effects require further research.

Author Contributions

Conceptualization, U.S. and M.M.; methodology, U.S., M.M. and M.D.; software, U.S. and M.D.; validation, M.M.; formal analysis, U.S., M.M., B.R. and M.D.; investigation, U.S., M.M., B.R. and M.D.; resources, M.M.; data curation, U.S.; writing—original draft preparation, U.S., M.M., B.R. and M.D.; writing—review and editing, U.S., M.M., B.R. and M.D.; visualization, B.R. and M.D.; supervision, M.M. and M.D.; project administration, M.M. and M.D. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal protocols used in this work were evaluated and approved by the II Local Ethical Committee on Animal Testing in Warsaw on behalf of the National Ethical Committees on Animal Testing (protocol code WAW2/089/2020 approved on 29 July 2020). They are in accordance with FELASA guidelines and the National law for Laboratory Animal Experimentation (Dz. U. 2015 poz. 266 and 2010–63–EU directive).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Tables

Table 1

Heart rate (HR) and lactate concentrations (LAC) measured during the following treadmill exercise sessions: dry treadmill (DT) session, water treadmill (WT) session at fetlock-depth water, WT session at fetlock-depth water with artificial river (AR) mode, WT session at carpal- depth water, and WT session at carpal-depth water with AR mode. Data are presented as median and ranges (minimum and maximum values).

	Time Point	DT Session	Fetlock-Depth WT Session	Fetlock-Depth WT +AR Session	Carpal-Depth WT Session	Carpal-Depth WT +AR Session	p-Value
HR	before	35 (28–50) a	38 (28–46) a	38 (29–50) a	36 (29–42) a	37 (30–45) a	p = 0.31
(bpm)	fin. 60 s	98 (80–105) b,X	106 (98–118) b,Y	103 (96–119) b,Y	111 (100–123) b,YZ	114 (101–136) b,Z	p < 0.0001
	30 min	72 (48–82) bc,X	52 (41–68) c,Y	56 (40–85) c,Y	69 (40–76) bc,XY	61 (41–76) c,XY	p = 0.002
	24 h	40 (32–45) ac	36 (28–44) a	35 (29–42) a	36 (60–42) ac	37 (30–46) a	p = 0.06
	p-value	p < 0.0001	p < 0.0001	p < 0.0001	p < 0.0001	p < 0.0001
LAC	before	0.5 (0.5–0.5) a	0.5 (0.5–0.5) a	0.5 (0.5–0.5) a	0.5 (0.5–0.5) a	0.5 (0.5–0.5) a	no var.
(mmol/L)	0 min	0.8 (0.5–1.8) b,X	0.8 (0.6–1.3) b,XY	1.3 (0.8–1.6) b,YZ	1.4 (0.7–2.2) b,Z	1.5 (1.1–2.9) b,Z	p < 0.0001
	30 min	0.5 (0.5–0.5) a,X	0.5 (0.5–0.5) a,X	0.5 (0.5–0.8) a,XY	0.6 (0.5–1.7) a,XY	0.7 (0.5–0.8) bc,Y	p < 0.0001
	24 h	0.5 (0.5–0.5) a	0.5 (0.5–0.5) a	0.5 (0.5–0.5) a	0.5 (0.5–0.5) a	0.5 (0.5–0.5) ac	no var.
	p-value	p < 0.0001	p < 0.0001	p < 0.0001	p < 0.0001	p < 0.0001

Lowercase letters (a, b, c) indicate differences in physiological responses measures across time points supported with p-value presented in the column. Capital letters (X, Y, Z) indicate differences in physiological responses measures across DT/WT exercise sessions supported with p-value presented in the row. No var.—no variability. Statistical significance was set at p < 0.05.

Table 2

Red blood cell count (RBC) and hemoglobin concentration (HGB) measured during the following treadmill exercise sessions: dry treadmill (DT) session, water treadmill (WT) session at fetlock-depth water, WT session at fetlock-depth water with artificial river (AR) mode, WT session at carpal-depth water, and WT session at carpal-depth water with AR mode. Data are presented as median and ranges (minimum and maximum values).

	Time Point	DT Session	Fetlock-Depth WT Session	Fetlock-Depth WT +AR Session	Carpal-Depth WT Session	Carpal-Depth WT +AR Session	p-Value
RBC	before	7.6 (6.5–9.1)	7.9 (7.3–9.2) a	7.4 (6.4–9.3) a	7.7 (6.6–9.3)	7.6 (6.8–9.1) a	p = 0.12
(×1012/L)	0 min	8.9 (6.4–9.9)	7.5 (6.7–9.4) a	7.8 (6.7–10.0) a	7.3 (6.2–9.8)	7.6 (6.7–9.9) b	p = 0.89
norm: 2–12× 1012/L	30 min	7.3 (5.4–8.6)	7.4 (5.7–9.1) b	7.3 (6.3–8.0) b	7.1 (6.0–8.7)	7.0 (5.9–8.0) a	p = 0.18
	24 h	7.4 (6.9–8.8) X	7.0 (5.9–8.5) b,XY	6.9 (5.9–8.1) b,Y	7.6 (5.6–9.6) XY	7.4 (6.3–8.5) a,Y	p = 0.009
	p-value	p = 0.06	p = 0.007	p = 0.0003	p = 0.11	p = 0.01
above N/n:		0	0	0	0	0
HGB	before	11.4 (10.4–12.3) a	12.6 (10.4–12.3) a	11.7 (10.2–12.8) ab	11.8 (10.3–13.4)	11.5 (10.3–14.2)	p = 0.07
(g/dL)	0 min	13.8 (10.1–16.2) b	12.1 (10.9–14.3) a	11.9 (10.7–15.6) a	12.1 (10.8–14.6)	12.2 (10.1–14.6)	p = 0.17
norm:8–17 g/dL	30 min	11.6 (8.7–12.9) a	11.4 (10.0–14.0) b	11.4 (9.3–12.8) b	11.5 (9.9–12.7)	11.1 (10.2–13.7)	p = 0.62
	24 h	12.2 (11.3–14.8) a,X	11.3 (10.0–13.8)b,Y	11.0 (9.8–13.1) b,Y	12.2 (9.6–14.6) XY	11.9 (10.7–15.0) XY	p = 0.006
	p-value	p = 0.002	p = 0.003	p = 0.008	p = 0.12	p = 0.09
above N/n:		0	0	0	0	0

Lowercase letters (a, b) indicate differences in physiological responses measures across time points supported with p-value presented in the column. Capital letters (X, Y) indicate differences in physiological responses measures across DT/WT exercise sessions supported with p-value presented in the row. Norm—the normal reference ranges; above N/n—the number of measures (N) and the number of horses (n) with a hematological parameter value above the normal reference range. Statistical significance was set at p < 0.05.

Table 3

Creatine phosphokinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) measured during the following treadmill exercise sessions: dry treadmill (DT) session, water treadmill (WT) session at fetlock-depth water, WT session at fetlock-depth water with artificial river (AR) mode, WT session at carpal-depth water, and WT session at carpal-depth water with AR mode. Data are presented as median and ranges (minimum and maximum values).

	Time Point	DT Session	Fetlock-Depth WT Session	Fetlock-Depth WT +AR Session	Carpal-Depth WT Session	Carpal-Depth WT +AR Session	p-Value
CK	before	204 (79–279) a	180 (127–240) a	203 (89–275) a	157 (103–263) a	155 (109–222) a	p = 0.06
(U/L)	0 min	214 (116–282) ab,X	267 (167–397) b,Y	276 (176–361) b,Y	249 (200–388) b,XY	270 (170–440) b,Y	p = 0.005
norm: 90–565U/L	30 min	243 (181–454) ab	234 (137–479) ab	232 (133–471) ab	250 (183–420) ab	275 (190–448) ab	p = 0.79
	24 h	260 (169–482) b	220 (117–348) ab	248 (140–346) ab	231 (117–367) ab	241 (116–334) ab	p = 0.08
	p-value	p = 0.001	p = 0.003	p = 0.02	p = 0.0002	p < 0.0001
above N/n:		0	0	0	0	0
AST	before	292 (194–394) a	262 (214–362) a	301 (210–373)	281 (206–421)	280 (189–298) a	p = 0.31
(U/L)	0 min	317 (272–423) b	332 (267–415) b	311 (262–449)	309 (248–440)	348 (272–486) b	p = 0.16
norm:1–450 U/L	30 min	347 (290–425) b	357 (274–443) b	324 (252–445)	317 (240–444)	383 (239–492) b	p = 0.06
	24 h	326 (297–439) ab	390 (293–450) b	316 (256–429)	311 (275–436)	393 (266–482) b	p = 0.12
	p-value	p = 0.005	p < 0.0001	p = 0.42	p = 0.09	p = 0.001
above N/n:		0	0	0	0	5/3
LDH	before	380 (292–489)	329 (214–502) a	440 (230–505)	352 (260–471)	423 (324–581)	p = 0.06
(U/L)	0 min	431 (241–708) X	424 (246–599) ab,XZ	398 (236–497) Y	397 (234–705) XY	482 (335–816) Z	p = 0.001
norm:0.1–639 U/L	30 min	398 (224–718) XY	506 (316–646) b,X	402 (213–603) Y	411 (240–725) XY	494 (292–691) X	p = 0.002
	24 h	415 (227–493) XY	495 (250–719) b,X	327 (227–559) XY	388 (229–517) Y	455 (291–629) XY	p = 0.007
	p-value	p = 0.05	p = 0.0008	p = 0.26	p = 0.23	p = 0.06
above N/n:		2/1	2/2	0	2/1	3/3

Lowercase letters (a, b) indicate differences in physiological responses measures across time points supported with the p-value presented in the column. Capital letters (X, Y, Z) indicate differences in physiological responses measures across DT/WT exercise sessions supported with p-value presented in the row. Norm—the normal reference ranges; above N/n—the number of measures (N) and the number of horses (n) with a biochemical parameter value above the normal reference range. Statistical significance was set at p < 0.05.

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