Four Weeks of Incline Water Treadmill Exercise Can Contribute to Increase Epaxial Muscle Profile in Horses

Background Water treadmill (WT) exercise is a popular modality for the training and rehabilitation of horses. However, evidence-based literature regarding the use of WT exercise, particularly using inclines, is lacking. Objectives The aim of this study was to assess the effect of recurring inclined WT sessions on equine epaxial muscle development. Methods Six horses completed 24 sessions of 15 minutes of WT activity over four weeks. Horses walked with water at the midcannon level at a treadmill incline of 4%. Back traces were measured at three and seven centimetres ventral to the dorsal midline at T5, T9, T14, and T18, prior to the first session (W0) and weekly for 4 weeks (W1–4). Results Overall, the back traces demonstrated progressive increases in muscle development (p < 0.05), starting at W2 up to W4. At three centimetres ventral to the dorsal midline, the most to least significant increases in gross muscle development were at T18, T5, T9, and T14, respectively, and when measured at seven centimetres ventrally, the most to least significant increases were demonstrated at T5, T18, and T14. It was noted that increases in thoracic back profile musculature were mainly observed within two to four weeks of the WT intervention. Conclusions It has been concluded that repeated WT exercise on an inclined setting has a significant effect on the rate and size of growth of equine thoracic back profile musculature. Muscle hypertrophy due to resistance training in the WT starts at 2 weeks within the programme, and it progresses as exercise continues to be performed.


Introduction
Water treadmill (WT) is a popular exercise for training and rehabilitation of horses [1].Most of the research studies on equine WT are focused on the efects of the exercise during its practice, not on the long-term efects that can be accumulated.It has been well established that walking on a WT at diferent water levels will elicit diferent kinematics on limbs and back [2][3][4][5][6][7].Although WT are used for rehabilitation of limbs and back injuries [1,8], there is currently a paucity of data regarding the long-term efect of WT exercise, particularly whether the use of water treadmill (WT) exercise on an inclined setting causes an increase in musculature over the back of horses.It is however understood that due to the unnatural movement of the treadmill belt causing the horse to become unbalanced they then chose to employ the neck and back muscles in order to re-establish their natural stride pattern.Once accustomed to the UWTM, the horse can freely engage the muscles along their back without the added weight of a rider or saddle on top; the weight of the saddle and rider combined has been previously found to have a signifcant negative impact on the thoracolumbar posture [8].It has also been observed that as the water level increases from the baseline, there is a greater amount of fexion caused over the lumbar region of the back [3,4].Interestingly, the velocity of the treadmill belt is also said to have an infuence on the fexion and extension of the spine; as the velocity of the treadmill belt increases, the horse's body recruits the main muscles known for stabilising the vertebral column (m.longissimus dorsi and m. rectus abdominis) [9].Te m. rectus abdominis works as an antagonist muscle to the m.longissimus dorsi for stabilisation in the horse, and so, with the increasing water level, this muscle works to fex the thoracolumbar spine; it could be hypothesised that this will in turn have a cumulative efect on the muscle growth over the back profle of the horse [10].Te bow and string theory can be used to help visualise the fexion and extension of the horses' back; there are three main biomechanical observations of the equine back during locomotion known as the planes of movement: pelvic fexion (PF), lateral bending (LB), and axial rotation (AR) [5].Te bow and string theory relates with the fexion and extension of the thoracolumbar spine caused by the stabilisation muscles m. longissimus dorsi and m. rectus abdominis working antagonistic to one another.Te m. longissimus dorsi's eccentric and concentric contractions are synchronised with the opposing contractions of the m.rectus abdominis to maintain the centre of mass over the base of support [11] in order to counteract the destabilising forces caused by the internal visceral mass during ambulation [12,13].However, it is important to remember that every case is diferent, and an increase in thoracolumbar fexion is very much dependent on the horses' spinal conformation and range of motion (ROM) of their spinal joints [8].Previous studies carried out on ventral spinal fexion and extension in walk and trot have shown a linear increase in the muscular electromyographic activity of both the m.longissimus dorsi and m. rectus abdominis when changed from an incline of 0% to 6% [13,14].It could therefore be postulated that the recruitment of the stabilisation muscles during walking on an UWTM whilst on an inclined setting should in fact show an increase in facilitation and development of muscle across the thoracolumbar back profle more than walking on a levelled surface would.Remarkably, after just 10 days, UWTM training was shown to afect equine back ROM [5] Tere are, however, little to no studies which have been carried out that describe or, in fact, assess the efect that UWTM training has on the equine back profle musculature; further research studies such as the current study are needed in order to provide an insight into this gap in knowledge.Terefore, the aim of this study was to assess the efect of recurring inclined WT sessions on equine epaxial muscles' development.We hypothesised that the inclined WT therapy would increase muscle mass in the epaxial musculature.

Materials and Methods
Te material in this manuscript has been acquired according to the guidelines set by the Animal (Scientifc Procedures) Act 1986 and the Declaration of Helsinki and has been approved by the Animal Welfare and Ethics Committee of Writtle University College.Te approval number is 98360534/2019.A written informed consent was obtained from the owners of the participants of the study.Veterinary surgeon consent was obtained.

2.1.
Horses.Tis study involved six healthy, clinically sound, and WT-habituated horses, with a mean ± standard deviation (SD) age of 9.5 ± 3.73 years old, and the mean height was 169 ± 22 cm.Te six subjects encompassed four breed types as follows: Hanoverian (n � 1), Toroughbred (n � 1), Irish Sport Horse (n � 3), and Dutch Warmblood (n � 1).All six were sourced in Northern Ireland from the Irish Equine Rehabilitation and Fitness Centre.Horses were training and competing at varying levels mainly for dressage and showjumping.Horses were stabled at the Irish Equine Rehabilitation and Fitness Centre, and each of them was fed their own standard diets for the duration of the trial.Besides the WT exercise, horses had a walk in-hand exercise, ranging from 5 to 10 minutes a day at the start of the trial, reaching 15 to 20 minutes a day by the end of the trial.To ensure validity and reliability of the research, the same experienced handler prepared, loaded, and secured each horse in the treadmill and operated the individual treadmill programmes.Te sample size was calculated using the resource equation approach [15], based on published studies assessing back musculature increase as cross-section area with diferent therapies, and adequate statistical discrimination has been achieved between 3 and 8 horses per group [16,17].Assuming that the methods difer in their intervention and outcomes, estimates by 10%, for Type I and II errors of 0.05 and 0.20, respectively, using the Bland-Altman test, we estimated a sample size between 5 and 11 horses would be needed (MedCalc ® Statistical Software version 20.115).

Water Treadmill (WT) Exercise. Te WT (ECB Aqua
Treadmill, ECB Equine, UK) sessions were standardised for all horses and weeks and consisted of 24 sessions of 15minute each over four weeks, with 6 sessions being undertaken weekly.Te previous measurements were called W0, the measurements after the frst week were named W1, and so on until the measurements after the fourth week of exercise, week 4, which were named W4.Te water height, on every session, was the middle of the third metacarpal bone.Te WT belt speed was determined by selecting a safe but also efective walking velocity that was comfortable for each horse, as assessed by the therapist, which was then maintained throughout the entirety of the data collection.Speed ranged from 3.5 to 3.8 km/h with a mean of 3.63 ± 0.14 km/h.Angle of incline for the WT belt was set at 4% to ensure all subjects could cope with the same inclination intensity.

Back Profle Dimension Measurements.
Prior to the frst WT treadmill session, ffth (T5), ninth (T9), fourteenth (T14), and eighteenth (T18) thoracic vertebrae were identifed by palpation.Measurements were taken at each location using a saddle ftting kit (the Perfect Fit, San Francisco, USA) which includes a 50 cm fexible curve ruler (pliable metal encompassed by rubber) to shape around the dorsal surface of the back, following the contours of the body (Figure 1).A fexible curve ruler can be moulded to the contours of the spine and has been validated for assessment of transverse muscular profle [18].
Te levels identifed for the relevant vertebrae were marked with clipping a small patch of hair to ensure the measurements were repeated at the same landmarks.Te subsequent outline was then mapped out on A3 graph paper (Figure 1) [18] for each timepoint on a separated sheet to denote the diferent measurements and avoid bias.For one single horse, besides tracing the back profles of each timepoint on separated sheets, the profles were also traced 2 Veterinary Medicine International on a common sheet to allow the visualisation of all timepoints on single sheet, only for purposes of illustration of results.Te same researcher (NF) repeated each measurement throughout the study.Before taking measurements, the assessor ensured the horse was standing squarely, with its weight evenly distributed over all four limbs, and the head and neck positions were maintained in a neutral posture with the horse's mouth level with the point of the shoulder by an assistant.All measurements were taken in a concrete fat surface adjacent to the WT.Te thoracic back-profle dimension was measured immediately before the frst WT session (W0) and after each 6 sessions (weekly W1-W4), totalling 5 measurements during the 4 weeks of data collection.Te measurements after sessions was carried out 20 minutes after the exercises to ensure cool down, as it has been reported that immediately after exercise, the muscle cross-sectional area may change [19].Te thoracolumbar dimensions were defned as the widths, in cm, at two levels: 3 cm and 7 cm ventral to the dorsal midline (Figure 2).

Statistical Analysis.
Once all measurements were taken, a standard ruler was used to measure the width of the back traces, in centimetres, at three and seven centimetres below the dorsal line, using each of the A3 traces collected, with data recorded in excel, and subsequently transferred to SPSS v26.0.Te mean ± SD of the back traces at each timepoint was calculated and subjected to the Shapiro-Wilk test for normality which showed parametric data.Following this, all data underwent Mauchley's test of sphericity to identify whether the assumption of sphericity had been violated, if this was the case, the Greenhouse-Geisser correction was applied to the within subjects' efects.From here, it was ascertained as to whether the back traces exhibited statistically signifcant increases (p < 0.05) between diferent timepoints.As all the data were parametric (p > 0.05) (apart from W7, M5 on T14; however, there were no outliers as assessed by inspection of the boxplots), they all underwent assessment by the means of one-way repeated measures analysis of variance (ANOVA).A post hoc analysis was then conducted with Bonferroni correction (95% confdence interval (CI)) assessments in order to determine where the statistically signifcant diferences of the back traces lay and between which timepoints.Te results report SPSS Bonferroni adjusted p values.

Results
An example of back-width measurements taken over the period of four weeks can be seen in a representative image of the back-profle dimensions measurement which is presented in Figure 2 (note that this is just an illustrative fgure, and the measurements were taken on timepoints traced in separated sheets to avoid bias).

Discussion
Te present study was conducted with the purpose of investigating the efcacy of recurring WT exercise with inclined resistance to cause increases in thoracic epaxial muscles of horses.Overall, signifcant increases in epaxial muscle profle have started to be shown at W3 of the WT therapy, with usually a further increase at W4. Te most signifcant changes were seen at T5 and T18 levels, although all back points assessed have shown some increase in epaxial muscle profle.Our results, as a whole, have supported the hypothesis that WT therapy can help on the development of the epaxial muscles.To the authors' knowledge, the present study is the frst of its kind to periodically measure the diference in equine back profle musculature over a fourweek period, using back traces at four diferent points along the thoracic vertebrae.Current literature regarding the use of the WT as a rehabilitation tool is limited with respect to its efcacy in activating and building the paraspinal musculature in the thoracic region.
Tis study demonstrates statistically signifcant increases in muscle development at each of the anatomical reference points (T5, T9, T14, and T18) at three centimetres ventral to the dorsal midline, with the most signifcant increases in muscle development noted at T18.Although during UWT work, the thoracolumbar joint (T18/L1) sees a decrease in fexion-extension in comparison with more cranial thoracic areas [2], we could infer that deeper stabilising muscles may have been activated.It is well known that inclination of treadmill plays a role in epaxial muscle activation, particularly longissimus dorsi [13].When treadmill inclination changed from 0 to 6%, EMG activity of the longissimus dorsi began and ended later; therefore, a longer activity duration was noted [13].Lower inclinations such as 3% has also been reported to increase EMG values for the longissimus dorsi [20], and this efect of the incline has contributed Veterinary Medicine International towards the increased epaxial profle reported in our study, as we have used an incline of 4%.Te next most statistically signifcant increases in muscle growth were found at T5, noted as the highest point of the withers, and as such is subject to the greatest level of shear forces during ridden exercise due to direct transmission of rider's weight through the stirrups, which may afect the muscles responsible for protraction and retraction of the forelimbs [21].It could be suggested that the muscle development in this region may be due to the horse being able to freely engage the muscles in this area without the negative infuence of a rider or saddle during the WT exercise [4].In addition, it could be postulated that the increases in size of the musculature were facilitated by the water level being set low enough to allow the horses to lower their head and neck, causing traction on the withers from the nuchal and supraspinous ligaments [22,23].Tis creates cranial thoracic fexion [24] and activates the vertebral stabilisation muscles.Furthermore, research has reported that WT exercise increases considerable activation of m. splenius [20], and as this muscle inserts at T5, its activations during WT exercises could have contributed towards the muscle profle observed at this level in our study.
Statistical signifcance was also apparent at T9 between diferent timepoints, which may be associated with the specifc anatomical characteristics of the vertebrae involved in the adjacent articulations, which determines the degree of movement at each joint complex [25].Te body of vertebral segments T2-T9 is shorter but provides larger points of attachment for the nuchal and supraspinous ligaments as well as for the relevant muscles which produce the movements of the back [26].Terefore, T2-T9 possess greater amounts of intervertebral movement, displacement, and axial rotation between these joint complexes, due to the elasticity of the ligaments [23,25,26].Te aforementioned biomechanical characteristics of the ninth thoracic vertebrae may have contributed to muscle build by allowing increased spinal ROM, stimulating antagonistic epaxial and hypaxial muscle contractions to resist excessive displacement of the vertebral column [27].Furthermore, it has been discovered that movements of the back, limbs, head, and neck are closely associated [25,28]; therefore, it can be assumed that exercising on an incline will require synergistic contractions of both the back and hindlimb muscles to produce propulsion in the sagittal plane [12].
We therefore hypothesise that the benefts of the incline associated with the benefts of water at midcannon bone have created a synergic beneft for the muscular development in our study.
Te vertebral segment which revealed the least diferences in muscle build between the various timepoints was T14, only demonstrating a statistically signifcant average increase of 2.9 cm between week one and four of WT intervention.Upon closer inspection of the results, apart from an initial decrease following the frst week of WT exercise, it is clear that a smaller cumulative increase in gross muscle size occurred each week; however, these may have been too minimal to reach statistical signifcance.Te statistically signifcant increase in muscle mass between week one and four may have occurred as the greatest amounts of axial rotation and lateral bending occur at the vertebral level of T14 [26,29] due to the presence of asternal ribs, which are indirectly attached to the sternum [26].Hence, the increased axial rotation and lateral bending experienced in this vertebral region, along with the increased dorsoventral fexionextension caused by exercising on an incline [12,14,30], are both expected to have infuenced the facilitation and therefore also the gradual increase in mass of the m.longissimus dorsi and m. rectus abdominis in the current study as they are required for vertebral stabilisation in the horse [30].What is worthy of note is that the timing of increases correlate with the efect of resistance training and the process of hypertrophy, whereby a minimum of two to four weeks of resistance training are required in humans to facilitate skeletal muscle hypertrophy [31].
At 7 cm, our results indicated that the most signifcant increases in overall thoracic musculature development occurred at T5 and T18.A possible explanation for the differences noted between the back traces at three and seven centimetres could be attributable to the function of the hypaxial muscles which are situated ventrally to the transverse processes of the spine.Te hypaxial muscles mainly function to produce fexion of the cranial thoracic spine, while the m.rectus femoris is activated to counter spinal extension [32].Furthermore, as the m.rectus abdominis inserts onto the head of the femur, it could be postulated that this muscle plays a role in the synergistic contractions of the back and hindlimbs during forward propulsion [12] possibly explaining the signifcantly increased muscle mass in the region.
Comparatively, the next most statistically signifcant increases in thoracic muscle development were observed at T18.It could be inferred that the increased muscle growth observed when measured further ventrally could be linked to the slightly more lateral and ventral origin of the m.obliquus abdominis externus on the lateral surface of ribs 4-18 and the thoracolumbar fascia.Likewise, the m.rectus abdominis is recruited during the second half of the stride in the region of T18 once the forelimb is protracted and ipsilateral hindlimb is retracted to counter the excessive thoracolumbar extension caused by the downward movement of the internal visceral mass [27], possibly further explaining the increase in muscle development when exercising against increased resistance from both the water [7] and inclination [13,14].Statistical signifcance was also evident at T14, although not to the same extent as T5 and T18 as only two of the timepoints demonstrated increases in gross muscle development.Tis could also be attributable to the more controlled exercise in the sagittal plane without the added weight of a saddle or rider [4,33] which may allow for more symmetrical muscle development than when exercising on land [14].As for the signifcant increases in thoracic musculature observed at T14, the greatest amounts of axial rotation and lateral bending occur in this region during locomotion [26,29].Moreover, the horse also shows increases in hindlimb ROM when walking in water [7].Research by Mooij [5] has also suggested that in order to produce hindlimb movement in water, there must frst be Veterinary Medicine International a further increase in axial rotation of the caudal thoracic spine, which may explain the resulting increased muscle development at the level of T14.
No statistically signifcant increases in thoracic backprofle musculature were observed at T9 when measured at 7 cm; however, there was evidence of cumulative weekly increases in the back-trace widths when compared to the individual means.
Overall, our research agrees with a study recently presented, which performed a semiqualitative analysis of muscle development in horses undergoing WT work and has concluded that WT can increase thoracic epaxial muscle development, after 20 weeks of exercise [34].However, our study has found earlier improvements in muscle development at thoracic epaxial musculature.We attribute this earlier muscle development to the fact that we have used an incline on the WT, while the previous study [34] did not mention incline so was probably performed under fat WT.As mentioned before, both water and incline have an infuence on epaxial musculature activation and, therefore, growth.Hence, we believe that the combination of these two elements have contributed to the earlier positive results observed in our study.
Tat being said, our study also had its limitations.Te frst could be small sample size, which could lead to the assumption that our results may lack ecological value; that being said, the majority of our results demonstrated statistical signifcance between diferent timepoints, which warrants further investigation with a larger sample size in the future to more precisely evaluate the efects of the intervention, to allow more defnitive conclusions to be drawn.A potential limitation concerning the repeated measures study design is the efect that the treatment intervention has on subsequent treatments, the lack of a control group and not having fat UWT and inclined dry treadmill groups to ascertain which independent variable (water incline) was more relevant in eliciting the observed changes, or if both interventions will contribute towards muscle hypertrophy.As this study utilised a longitudinal layout over the course of four weeks, it could be said that biased estimates of the treatment efcacy may be made due to the carry-over efect of the treatment modality.However, this study design emphasised comparisons within each horse over time, and as horses had a similar trend on muscular growth, we can attribute this to the exercise on the inclined WT with a good certainty, although we cannot attribute the efects to the water or incline separated.Despite all limitations, it is our belief that the current study presents an insightful account into the rate and size of paraspinal muscle growth in the thoracic region through the implementation of repeated WT exercise with incline resistance.Another limitation is that as a result of being reshaped repeatedly, the fexible curve ruler is susceptible to deterioration over time; therefore, it is advised to frequently replace the fexible curve ruler used for data collection to ensure the results remain consistent.We have used the same piece of equipment all throughout the measurements, and we have not noticed any signifcant misshapen of the ruler.We also acknowledge that there are other more objective means to measure epaxial muscle changes in size as evaluation of cross-sectional area with ultrasound scans as described in the literature [16,35].However, our choice to use the fexible curve ruler was due to equipment availability and the fact that there is a validation for the use of this tool 9 .

Conclusions
Te results of this study support the original hypothesis, revealing that repeated WT exercise on an inclined setting does in fact have a signifcant efect on the rate and size of growth of the equine thoracic back profle musculature, although further research should be conducted to determine which intervention is more relevant, incline or water, and if both interventions have a synergic efect, as well as which specifc muscles are recruited during WT intervention with incline resistance and to establish the increases in the crosssectional area of the individual muscles with ultrasound scans rather than just the total increases in overall muscle development.

Figure 1 :
Figure 1: Representation of back profle dimension measurements at the T5 level.(a) Te fexible ruler was used at the T5 level, over the spine, to shape the back profle.(b) Te back profle was transferred to a graph paper.

Figure 2 :Figure 3 :Figure 4 :
Figure 2: Example graphs of the changes in back dimension before the water treadmill programme (W0) and after each week of treatment (W1-W4) of water treadmill programme at ffth (T5), ninth (T9), fourteenth (T14), and eighteenth (T18) thoracic vertebrae before.Te orange lines show the levels of measurement at 3 cm (orange line) and 7 cm (green line) ventral from the dorsal line.

Figure 5 :Figure 6 :
Figure 5: Muscle dimensions at T14 before the water treadmill programme (W0) and after each week of treatment (W1-W4) (n � 6).Standard error is shown.Statistically signifcant diferences, by repeated measures ANOVA, are indicated by diferent letters.Lower case letters are used for measurements of 3 cm ventral to the dorsal line.Capital letters are used for 7 cm ventral to the dorsal line.