Effects of aerobic , anaerobic , and concurrent training on bone mineral density of rats

A sample of 39 rats were divided into the following groups: baseline (BL); aerobic training (ET4 and ET8); anaerobic training (ST4 and ST8); and concurrent training (CT4 and CT8). The aerobic training was performed by swimming with a load corresponding to 70% of the anaerobic threshold; the anaerobic training was performed by jumping in water with a load corresponding to 50% of body weight; and the concurrent training combined the two protocols. The analysis of BMD was performed on the tibia of the animals and showed an increase in the density of the ST4, CT4, ET8, ST8, and CT8 groups when compared with both the BL and ET4. Thus, it is concluded that anaerobic exercise was shown to be effective in increasing the BMD of animals after four weeks of training; however, aerobic training was only able to raise BMD following eight weeks of training.


Introduction
Physical training is important for maintaining bone mass as it acts by means of mechanical and metabolic stimuli, modeling the synthesis of the bone matrix 1 .However, training presents variations in method of application with differences in duration and intensity that promote different stimuli, both mechanical and metabolic 2 .
Among the various classifications of physical training, aerobic and anaerobic are highlighted and defined according to their metabolic dominance.Aerobic training presents a low-intensity and long-duration, differing from anaerobic training, which is characterized by a high-intensity and short-duration 3 .
Aerobic training is widely used for the treatment of osteoporosis as it presents a low-risk of fractures due to the low-intensity combined with metabolic stimulus necessary for bone synthesis, while anaerobic training presents significant mechanical stimulation due to its high-intensity 4 .
Increased bone synthesis increases bone mineral density by increasing synthesis of hydroxyapatite crystals in the bone matrix 1,2 .The method used to quantify bone mineral density (BMD) is dual energy X-ray absorptiometry (DXA), which quantifies the mineral content of the bone 2,5 .
Concurrent training was conducted with the two exercise modalities -aerobic and anaerobic -in the same training session.The effects of this type of training present disagreement in the literature with studies that show beneficial effects, harmful effects, and no effects 3,6 .
The use of concurrent training could promote increased bone mineral density, combining the effects of aerobic and anaerobic training and serve as a resource for new interventions in populations at risk of diseases, such as osteoporosis, and could assist in growth phases during which bone tissue develops in children and young people 3,6,7 .
Therefore, the present study aimed to evaluate the effects of aerobic, anaerobic, and concurrent training on the bone mineral density of rats.

Sample
In total, 39 rats were used (Wistar).The animals were 100 days' old and obtained from the Central Animal Laboratory of Unesp, Botucatu -SP.They were kept in the small animal vivarium at the Histology and Histochemistry Laboratory, Physical Therapy Department, Faculty of Science and Technology -FCT/UNESP, Presidente Prudente Campus -SP.The animals were placed in collective cages with up to five animals per cage (polyethylene) with an ambient temperature (22 ± 2°C), 12 hr light/dark cycle, humidity of 50 ± 10%, and were fed standard chow and water ad libitum.
All procedures were previously approved by the Ethics Committee on Animal Use of FCT/UNESP under protocol number 002/2011 in accordance with regulations established by the Brazilian College of Animal Experimentation (COBEA).

Experimental design
The experiment began with a determination of the training load through the Critical Workload test (CL).After defining the loads, the animals were submitted to aerobic, anaerobic, or concurrent training, and were euthanized after the training period.The animals of the BL group were euthanized immediately prior to initiation of the experimental period.

Critical Workload Test
Determination of the aerobic capacity, by means of the critical load, was obtained by inducing exercise in four different stimuli, which were randomized with loads of 7%, 9%, 11%, and 13% of the body weight so that all animals performed the four efforts and reached exhaustion after between two and ten minutes, as proposed by Hill 8 .However, due to the failure of the animals at the 13% body weight stimulation, this intensity was excluded from the determination of the anaerobic threshold.
The time taken to perform the exercise at each load was timed using a stopwatch (TIMEX®, model 85103).Every effort was performed at intervals of 24 hours, following the method of Chimin et al. 9 .Next, a linear regression was performed between the load and inverse of the time for each effort (1/tlim), from which an equation was generated to verify the value corresponding to the aerobic capacity, represented by the variable b in the equation (Figure 1).

Aerobic training protocol
For the aerobic training, the animals were submitted to 30 minutes of swimming, three times a week, in appropriate tanks subdivided by PVC cylinders to individualize the lanes so that each animal trained individually.The intensity was equivalent to 70% of the anaerobic threshold (AT), set by the Critical Workload test 9 .

Anaerobic training protocol
The anaerobic training was composed of four sets of 10 jumps, three times per week, in a cylindrical PVC container specially modified for jumps in water (50 cm long) with an appropriate depth for the size of the animals (38 cm deep).Between each series of jumps, a 1-minute interval was established and verified by a stopwatch.The overload used corresponded to 50% of the body weight of each animal, accommodated on the anterior chest by means of a vest 10 .

Concurrent training protocol
The concurrent training combined the two physical training models mentioned above.The exercise sessions were performed in sequence (aerobic and anaerobic training) without an interval between them, three times a week, comprising 30 minutes of swimming (load set to 70% Lan) and four series of 10 jumps with an overload of 50% of the body weight of each animal 3 .

Collection and preparation of material
Following the experimental period, the animals were euthanized through an overdose of an association of ketamine hydrochloride and xylazine, intraperitoneally, following ethical principles in animal research.

Concurrent training bone
The right tibias of the animals were removed by surgical procedure and then immersed in saline solution and stored in a conventional freezer (-20°C) for subsequent DXA analysis.

DXA
To perform the DXA analysis, the tibias were first thawed at room temperature and then submitted to densitometry analysis in the DXA, DPX-Alpha model, LUNAR®, belonging to the Aracatuba Faculty of Veterinary Medicine -FMVA/UNESP, using special software for small animals 2,10 .
The DXA analysis was based upon the measurement of the following parameters: total area of bone; Bone Mineral Content (BMC) and the relationship between BMC and area; and denominated Bone Mineral Density (BMD).

Statistical Analysis
Data were analyzed using the Shapiro-Wilk test, which confirmed the Gaussian distribution of the data, after which oneway ANOVA followed by Tukey's post-test were used to verify the differences between the groups.The tests were performed using SPSS 17 software with an adopted significance level of 5% (p < 0.05).

Results
The analysis of the area (cm²) of the bones demonstrated lower values in the ET4 and CT4 groups in relation to the BL; there were no significant alterations between the other groups (Figure 2).Evaluation of the BMC (g) showed a significant increase in the ST4, ET8, ST8, and CT8 groups when compared to the ET4 group (Figure 3).The analysis of BMD (g/cm²) presented an increase in the density of the ST4, CT4, ET8, ST8, and CT8 groups compared with both the BL and ET4 groups (Figure 4).

Discussion
The results demonstrate that four weeks of aerobic training failed to increase BMD compared to the BL group, differing from the anaerobic and concurrent training, which significantly increased the BMD compared to the BL group.With respect to the eightweek period, all the training models significantly increased BMD in relation to the BL group, with no differences between the training methods in this period.However, no differences were observed between the anaerobic and concurrent training in either of the two training periods (ST4, ST8, CT4, CT8).Previous studies 2,11 have shown that the absence of mechanical stimuli, by means of immobilization or suspension of the animal, results in a reduction in BMD and fracture resistance.In contrast, physical exercise can act as an important non-drug intervention, increasing BMD and bone tissue resistance.Exercises with no impact, such as swimming and cycling, do not demonstrate an effect on improving BMD 12,13 ; however, studies that used swimming to reverse a BMD reduction framework caused by the absence of mechanical stimuli showed effectiveness in increasing BMD after swimming exercise 2,12,14 .
In the present study, although there was no difference between the ET4 and BL groups, the animals that performed the aerobic swimming exercise demonstrated increased BMD after eight weeks of training (ET8), compared to the BL group without intervention.Thus, swimming exercise, while not subjecting the bone tissue to impact, demonstrated an osteogenic effect, although of lesser magnitude since it was only able to increase BMD after eight weeks of training 12,13 .
Calcium deposition in bone is partly regulated by the mechanical stress to which the bone is subjected 2 .Although the mechanism involved in bone anabolism is not yet completely clear, it is known that a mechanical stimulus acts both in the inhibition of bone resorption through the action of NO (nitric acid), which promotes osteoclast inhibition, and in bone synthesis by increasing the intracellular calcium in osteoblasts and serum levels of IGF-1, factors that increase BMD and bone strength 15,16 .
In addition, exercise promotes metabolic and hormonal alterations that also stimulate the synthesis of calcium in bone tissue [15][16][17] .
Thus, the aquatic exercise-induced increase in mineral density (ET8 group) could be explained by the mechanical stress caused by the tendon traction on the bones during muscle contraction, together with the metabolic and hormonal alterations caused by exercise.
Therefore, it is suggested that this type of exercise can be used in conditions of severe loss of bone mass as a method of treatment, especially in cases with a high-risk of fractures, as the reduced impact on the bones from this exercise modality makes practitioners less susceptible to bone fractures 12,17 .
With respect to the anaerobic training, it presented higher osteogenic potential than aerobic training (swimming) since after just four weeks, it was able to significantly increase BMD with respect to the aerobic swimming training groups (ET4) and the group without training (BL), and the same occurred with concurrent training.In this way, the osteogenic effect of the water jump (anaerobic training) was demonstrated to be effective, even when combined with exercise without impact (aerobic swimming training) 18 .In addition, training with no impact was demonstrated not to be detrimental to BMD, since although it did not increase BMD, it also did not induce significant losses in the deposition of calcium in the bone, and did not detract from the osteogenic effect when combined with anaerobic jump training (concurrent group) 1,2,6 .
To increase BMD, exercises that provide impact on the musculoskeletal system are recommended, as calcium is deposited in bone tissue in response to tissue deformation.Hence, the greater the magnitude of the applied forces, the greater the stimulation of osteoblasts to synthesize calcium in the bone matrix 2,19 .Therefore, exercises with impact present a greater osteogenic effect, as in addition to alterations in the metabolism generated by exercise, they also act through mechanoreceptors, further stimulating bone synthesis 1,20 .
The present study was limited to evaluating the effects of training on bone tissue, although other factors also influence

Concurrent training bone
BMD, such as the condition of nutrition, these factors were not measured.Moreover, studies evaluating BMD in pathological conditions, such as osteoporosis, would be of great importance.

Conclusion
It is concluded that anaerobic exercise was shown to be effective in increasing the BMD of animals after four weeks of training; however, aerobic training (swimming) was only able to increase BMD after eight weeks of training.

Figure 1 :
Figure 1: CL scatter plot with real values of the test performed by one animal.In this case, the Lan corresponded to ≈5.76% of the BW.

Figure 2 :
Figure 2: Box-plot with values of area in cm².* Significant difference in relation to the BL group.

Figure 3 :
Figure 3: Box-plot with the values of BMC (g).# Significant difference in relation to the ET4 group.

Figure 4 :
Figure 4: Box-plot with the values of BMD (g/cm²).* Significant difference in relation to the BL group; # Significant difference in relation to the ET4 group.