Increased Total Serum Alkaline Phosphate Is Associated With Enhanced Bone Mineral Density in Dogs With Interval Exercise Training


 There are growing attention and interdisciplinary efforts for One Health to attain optimal health and well-being for humans and animals. Exercise has been suggested as a powerful intervention for health care and fitness management in humans; however, few studies have demonstrated the beneficial effect of exercise on dogs. The purpose of this study was to examine the effects of exercise training on heart rate (HR), bone mineral density (BMD), muscle volume (MV), and hematological and serum biomarkers in dogs. Six healthy beagles completed the interval treadmill exercise protocol, developed based on the FITT principle, two times a week for 12 weeks. For a physiological parameter evaluation, the mean HR value was analyzed using a Polar H10 system and software program. Quantitative computed tomography was used to determine BMD in the femur and vertebrae and MV in the thigh before and after exercise training. To perform hematological and serum biochemical parameter assessment, blood samples were analyzed at zero and 12 weeks of exercise. We show that interval exercise results in a normal HR response and no adverse behavioral and physiological effects on dogs. Exercise improves BMD in the femur (541.6 ± 16.7 vs. 610.2 ± 27.8 HA, p < 0.01) and increases serum total alkaline phosphatase (TALP; 68.6 ± 9.2 vs. 81.3 ± 17.2, p < 0.01), aspartate aminotransferase (23.5 ± 1.0 vs. 33.5 ± 1.6, p <0.01), and creatine kinase (114.8 ± 5.3 vs. 214.0 ± 20.8, p < 0.01) levels. There is a positive relationship between BMD and TALP (femur: r = 0.760, p = 0.004; vertebrae: r = 0.637; p = 0.025). Our findings suggest that long-term interval exercise training is beneficial to increase BMD in the femur, and an increased TALP level is a concomitant mechanism for enhancing BMD with exercise in dogs.


Introduction
Dogs, a common companion animal, have coexisted with humans since the Neolithic Age and are recognized as family members in many modern societies 1 . Studies have reported that over 70% of dog owners feel affection for their canine companions akin to raising a baby 2 . As the bonds between dogs and humans grow stronger, there is greater demand for better canine health care and tness management 1 . Exercise has been recognized as an essential component of "One Health", an interdisciplinary effort to attain optimal health and well-being for humans and animals 2,3 . Many human studies have robustly established that exercise improves health and tness [4][5][6][7] , but research exploring the bene cial effects of exercise in dogs is limited.
Studies have shown that physiological and biochemical parameters are fundamental to investigate the effects of exercise on health, tness, and function [4][5][6][7] . Heart rate 7 is a primary physiological indicator for diagnosing cardiac function and aerobic performance. Exercise-induced adaptation in HR (i.e., reduction in resting HR) is related to improved cardiovascular tness 6 . HR response during competitive exercise and recovery can also be used to assess heart-rhythm disorders, such as arrhythmia, in dogs 6 . HR levels in uence cardiac output and VȮ 2max during exercise 7 . Hematological and serum biochemical analyses can reveal systemic and metabolic functions. Treadmill exercise studies showed signi cant improvements in rectal temperature, glucose and lactate concentrations, red blood cell counts, hematocrit, and HR in dogs 8,9 . Although each parameter has its bene ts, comprehensive examinations are required for a thorough screening of a dog's health and tness.
Bone mineral density (BMD) and muscle volume (MV) are valuable measurements to assess overall health status and bone-muscle interactions in post-exercise dogs. Some studies have found that low BMD and MV are associated with several disorders, including in ammatory diseases, tumors, osteogenesis imperfecta, degenerative arthritis, and endocrine diseases 10,11 . Furthermore, exercise (e.g., sprinting, jogging, weightlifting, swimming) can be a powerful intervention for the prevention and treatment of these conditions in humans [12][13][14] . In several other studies, participants of different ages and genders showed positive changes in BMD, MV, and bone turnover serum biomarkers (e.g., bone-speci c alkaline phosphatase, deoxypyridinoline, and calcium) after exercise designed according to the Frequency, Intensity, Time/duration, Type, Volume, and Progression (FITT-VP) Principle [15][16][17][18][19] . To date, however, there has been little scienti c examination of the effect of exercise on bone and muscle health in dogs. One reason for this lack of knowledge is the ethical issue of studying bone and muscle tissues, which requires invasive procedures.
Non-invasive quantitative computed tomography (QCT) is a three-dimensional non-projection technique to evaluate BMD and MV. In human studies, QCT has been used to investigate muscle-bone interactions 20,21 . In veterinary medicine, however, QCT is mainly used to diagnose skeletal changes in osteoporosis and other metabolic bone diseases [22][23][24] . To date, there are no studies using QCT to assess changes in BMD and MV after exercise in dogs, nor have there been studies that examined the relationship between biomarker levels and bone-muscle properties. We postulated that it would be possible to use QCT to assess subtle changes in BMD and MV after treadmill exercise training. In this study, we modi ed a 12-week interval exercise protocol that yielded positive changes in physiological parameters for humans and applied it to healthy dogs 4 to explore the effects of exercise and associated mechanisms by identifying changes in HR, BMD, MV, and bone serum markers.

Results
Heart rate. Figure 1A shows the HR response during interval exercise, which includes a series of workout (W) stages and incomplete resting (R) stages. HR was analyzed for all beagles who completed the interval exercise protocol. Mean HR during the W stage was signi cantly higher than that during the R stage (p < 0.01). To evaluate if the exercise intensity protocol was progressively overloaded, we compared the mean HR of the W stage in every two protocol interval. The mean HR during protocols 7-12 was signi cantly higher than for protocols 1-6 ( Fig. 1B). Also, the mean resting HR was 83.8 ± 6.3 bpm, and the highest HR recorded was 232 bpm (data not shown).
Bone mineral density and muscle volume. BMD in the femur and vertebrae was measured using QCT before and after exercise. Post-exercise femoral BMD (610.2 ± 27.8 HA) increased signi cantly by 12.6% (p < 0.01) compared with pre-exercise BMD (541.6 ± 16.7 HA). In contrast, there was no difference in Page 4/17 vertebral BMD, although exercise (317.2 ± 6.6 HA; 291.4 ± 5.4 HA) increased BMD by 8.8%. Also, MV values before and after exercise were not signi cantly different (Table 1). Table 1 Analysis of bone mineral density and muscle volume before and after exercise in dogs. Bone mineral density and muscle volume data are represented as mean ± SEM. * Signi cant difference between pre-exercise and post-exercise (p < 0.05). The rate of increase in parameters was calculated as (post-exercise mean -pre-exercise mean) ÷ post-exercise mean × 100. Hematological and serum biochemistry parameters. Table 2 shows the differences in hematological and serum biochemistry parameters between pre-exercise and post-exercise. Levels of total alkaline phosphatase (TALP) as a serum bone marker showed a signi cant increase in post-exercise compared with pre-exercise (p < 0.01), but calcium and phosphorus levels were not different. Both AST and CK levels increased signi cantly after the exercise. All hematological and serum biochemistry parameters in preexercise and post-exercise dogs were within the reference range. Correlations between bone mineral density, muscle volume, and serum biochemistry parameters. Figure 2 illustrates the relationship between TALP and BMD in the femur and vertebrae and MV in the thigh. We found a signi cant correlation between TALP and BMD in the femur (r = 0.760; p = 0.004) and vertebrae (r = 0.637; p = 0.025). We also found a positive relationship between TALP and MV (r = 0.595; p = 0.041).
Those results provide evidence that exercise-induced increases in TALP are associated with increases in BMD and MV.

Discussion
The main objective of this study was to examine the effects of long-term interval exercise on HR, BMD, MV, and serum biochemistry parameters in healthy dogs. A primary nding was that the HR response to interval treadmill exercise in different stages and protocols was normal and a rmative. A secondary nding of our study indicates that interval exercise enhances BMD in the femur and increases TALP, aspartate aminotransferase, and creatine kinase biomarkers. To the best of our knowledge, these are the rst ndings indicating that long-term interval exercise training is feasible for dogs and improves BMD in the femur. We suggest that increased TALP levels may be an associated mechanism of increasing BMD with exercise in dogs.
Ferasin et al. 25 showed that dogs frequently refuse to exercise on the treadmill and are easily distracted in a laboratory environment. Due to those tendencies, adequate acclimatization is needed before initiating the exercise program. In this study, the beagles did not show any rejective or maladaptive behaviors. Also, the treadmill interval exercises did not cause any side effects or adverse reactions in healthy dogs. All dogs were able to complete the exercise program and were in good physical condition.
Our results are consistent with our previous ndings of normal physiological and behavioral responses to treadmill exercise 26 . This may be because the dogs had a su cient adaptive period on the treadmill, a well-designed exercise program was used, and adequate animal care was provided by the study veterinarians and researchers. Under these stringent experimental conditions, we aimed to explore the potential effect of long-term interval exercise on HR, BMD, MV, and serum biochemistry parameters in beagles.
The mean HRs of all dogs who performed interval exercise for 12 weeks had changed according to protocol intensity and progress. Also, following the FITT-VP principle, we were able to identify a regular HR change by organizing a suitable exercise program for dogs. Unlike other dog studies that found irregular HR patterns that were not proportional to the activity and external stimulus 27,28 , our results showed a normal HR response according to interval exercise, which may be because we created an optimal research environment by providing proper controls to anticipate the dogs' sensitivities to sounds and odors. The continuous and systematic HR change that we observed was within a maximum HR 230 bpm 7 during the interval exercise; this validates that the exercise program would be effective for improving cardiovascular health for dogs.
Next, we explored BMD and MV to evaluate whether an adaptive HR response to the exercise training protocol was bene cial to bone and muscle health. Our ndings corroborate previous evidence that interval exercise can increase BMD. The 12.6% increase in BMD that we observed is consistent with human studies that found an association between BMD and injury 29 or disease 30 . In humans, BMD is a key measure for diagnosing osteoporosis 30 , and a 3-5% increase in BMD has been shown to reduce fracture risk by 20-30% 30 . There are few studies of the effect of exercise on BMD in dogs and those studies found that aerobic exercise training in dogs resulted in either decreased or unchanged BMD 31,32 . In contrast, several human studies con rmed an increase in post-exercise BMD, regardless of age and sex 18 . The cause of this discrepancy is not clear, but the inclusion of intensity as a FITT component might be important because intensity induces a BMD increase and, thus, is a primary in uence on the extent of training effect 33 . Currently, the optimal intensity level for interval exercise to enhance BMD in dogs is not known 31 , but in humans, the proper endurance exercise intensity has been estimated to 55-75% of HR max 34 . In this study, the mean HR during the workout stage was 158.2 bpm, and the dogs continued to exercise for 36 minutes for twelve weeks. A previous study reported 230 bpm for HR max in their study dogs 7 ; thus, the intensity of interval exercise imposed on each dog in this study was approximately 68.7% of HR max . Furthermore, the combination of FITT components with progressive and overloading workouts may be associated with the BMD improvements observed in our study dogs.
The bene ts of regular exercise on BMD may be primarily linked to mechanical loading mechanisms 35 . Evidence for the Mechanostat Theory of mechanical loading has been con rmed in several animal studies 36,37 . Rats are tetrapodal animals that are known to have higher tibia stress because the tibia is subjected to greater weight-bearing during treadmill exercise compared with the vertebrae 38 . In a previous study of rats, treadmill exercise increased tibial BMD but not vertebral BMD 39 . Dogs, like rats, are tetrapodal animals, and their femurs are more likely to receive mechanical loads and to be more weightbearing than the vertebrae when running on treadmills 39 . Our ndings are consistent with the results of other animal studies and also support the concept that weight-bearing activity has a positive in uence on bone health 40 .
Many studies have suggested that treadmill exercise improves BMD, but the precise underlying molecular mechanism remains elusive 35 . Here, we examined the effects of treadmill exercise on serum bone markers such as calcium, phosphorus, and TALP to identify biological mechanisms. We found that exercise-induced increases in TALP are associated with increases in BMD. TALP is a critical biomarker to assess BMD accurately and e ciently in the absence of liver disease 41 . Several isoenzymes of TALP exist in various organs besides bone (e.g., liver, kidney), and serum TALP, derived mostly from bones, re ects the sum of those isoenzymes 42 . Particularly in young dogs, changes in TALP result from a bonespeci c isoenzyme 43 , because 96% of TALP consists of this bone-speci c isoenzyme 44 . The bonespeci c isoenzyme exists on the plasma membrane of osteoblasts and is carried through systemic circulation during the bone mineralization process 45 . TALP plays a role in the hydrolysis of inorganic pyrophosphate and then generates inorganic phosphate to maintain the appropriate ratio of inorganic pyrophosphate to inorganic phosphate, which is essential for the mineralization process 46 . Therefore, the upregulation of TALP in the two-year-old dogs from this study is considered a positive biomarker associated with increased BMD.
Also, correlation analysis revealed a positive association between TALP and MV in exercised dogs. To our knowledge, this is the rst report to identify a signi cant correlation between TALP and MV in exercised dogs. However, the cause of these consequences is not known, but exercise-induced crosstalk between muscles and bones may be involved. Further research is warranted.

Conclusions
We demonstrate that interval exercise has a positive impact on BMD in healthy dogs, and exerciseinduced enhancement of BMD is associated with increased TALP levels. Also, this study con rmed that QCT could be used as a measure to assess subtle changes in MV and BMD after a machine-running exercise intervention. Further investigations are needed to determine the impact of exercise on cardiovascular tness-, bone-, and muscle-related genes in dogs. Such research would improve our understanding of bone-exercise mechanisms and bone-muscle-interaction mechanisms, which would yield fundamental insights into key challenges in exercise science research.

Materials And Methods
Animals. Six healthy beagles were included in this study, and information on these dogs is provided in Table 3. All dogs were cared for following the recommendations described in The Guide for the Care and Use of Laboratory Animals. The study was approved by the Institutional Animal Care and Use Committee of Hanyang University and Seoul National University (2020-0073A, SNU-180731-2). All methods and protocols were carried out in accordance with the relavant guidelines and regulations. Before initiation of the experiments, baseline hematological analyses and body composition screening were undertaken by a veterinarian. Also, all beagles were subject to the same dietary and resting conditions. The dogs were housed in an environment with 12 hours (07:00-19:00) of bright light and 12 hours (19:00-07:00) of the dark. The temperature of the breeding room was 22ºC-23 °C, with 50-60% humidity. The dogs were kept in separate cages (775 x 960 x 900 cm) with soft rubber ooring that was cleaned daily. Meals were served twice a day (09:00, 17:00), and freshwater was provided freely. The dogs were not provided with food for four hours prior to exercise testing to prevent exercise-induced gastrointestinal distress, heartburn, and acid re ux. Weight (kg) 10.9 ± 0.5 Treadmill adaptation for dog safety. All dogs underwent two weeks of adaptive training to get acquainted with the researcher, laboratory environment, and exercise regimen in advance. The exercise training equipment included a treadmill (EGOJIN XG-V6E, Gyeonggi-do, Korea) and a safety belt, which was applied to each dog's chest. Rectal temperature was taken from each dog with a digital thermometer before and after exercise. Throughout the experiment, the researcher and veterinarian screened the dogs' behavior and con rmed their safety.
Interval exercise program. As a warm-up, the dogs performed a walking exercise for ve minutes at 2-3 km/hour prior to interval exercise. The exercise training program consisted of 12 treadmill protocols, which are detailed in Fig. 3. Each protocol was repeated twice per week for 12 weeks. The protocol consisted of a workout stage (W) and an incomplete resting stage (R). Exercise intensity was gradually increased by changing treadmill grade and speed.
Heart rate measurement. A Polar H10 HR measuring device and monitor (Polar Electro Oy, Kempele, Finland) were used to evaluate HR response during interval exercise. The dogs wore HR measuring devices on their chests, and HR data were collected every second. The mean HR value for each stage in all protocols was analyzed using the Polar Flow Software program (Polar Electro Oy, Kempele, Finland).
Quantitative computed tomography (bone mineral density and muscle volume). QCT was used to measure BMD and MV. All dogs were fasted for at least six hours prior to QCT scan. The dogs were intravenously premedicated with glycopyrrolate (Mobinul; Myungmoon Pharm., Seoul, Korea) at 0.01 mg/kg and then anesthetized with 6 mg/kg of propofol (Provive; Myungmoon Pharm., Seoul, Korea).
They were kept sedated with 1.5% iso urane (Foran solution; Choongwae Pharm., Seoul, Korea) and received 100% oxygen via endotracheal tube intubation. Percentage of oxygen saturation, end-tidal CO 2 , and HR were routinely monitored. QCT was performed using a 16-channel multidetecting CT scanner (Brivo 385; GE Medical System, Milwaukee, WI, USA). The lumbar vertebrae and femur were scanned with the dogs in dorsal recumbency. A calibrated QCT phantom (QRM-BDC/3; QRM GmbH, Moehrendorf, Germany) was placed under each dog. The scanning parameters were set as follows: 100 kV, 100 mAs, and 1.25-mm slice thickness and interval. The phantom and lumbar vertebrae were positioned parallel to each other. All QCT images were scanned using the bone algorithm. CT scan was performed before and after exercise (Fig. 1). All CT images were analyzed using commercially available software (RadiAnt DICOM viewer; Medixant, Poznan, Poland; Osirix DICOM viewer; Pixmeo, Geneva, Switzerland). The region of interest for QCT included only the vertebral body in the 3rd lumbar vertebra and was measured using an image at the origin of the transverse process. The cortical and trabecular bone at all measurement sites were included in the ROI. Femoral BMD was measured in the middle of the femoral neck, including one-third of the proximal diaphysis and one-third of the distal diaphysis. BMD was calculated from the CT image in Houns eld units. MV was measured at the correct position using multi planar reconstruction at the center of the femur, and a cross-section perpendicular to the bone was obtained. Each variable was measured three times, and the mean value for each was obtained.
Hematology and serum biochemistry parameter analysis. Blood samples for hematological and serum biochemical parameter analyses were collected the day before protocol one initiation and one day after protocol 12 completion (Fig. 4). Blood samples were kept in tubes coated with lithium heparin and stored at 4 °C. After blood withdrawal and plasma harvest, heparinized blood samples were allowed to clot and were then centrifuged to obtain serum. All analyses were performed within the rst six hours after blood extraction. Hematological parameters were measured from EDTA-blood samples using ADVIA 2120i (NYN Tarrytown, Tarrytown, NY, USA). Biochemistry parameters were measured from heparinized plasma using the Hitachi 7180 Auto analyzer (Hitachi, Tokyo, Japan) with reagents speci cally designed for the instrument.
Statistical analyses. All analyses were performed with GraphPad Prism 5.0 (GraphPad Inc., La Jolla, CA, USA). A one-way repeated analysis of variance was used to determine the mean difference in HR, followed by a Bonferroni post-hoc test. The mean difference in BMD, MV, and serum biochemistry parameters between pre-exercise and post-exercise was assessed using a two-  contributed equally as correspondence authors. All authors have read and agreed to submit the manuscript and declare that there is no con ict of interest. The results of the present study are presented ethically, without plagiarism, tampering, or manipulation by the researchers.

Data availability
All data used to support the ndings of this study are included within the article. The analyzed data during the current study are available from the corresponding author upon reasonable request.