Reduced bone formation and increased bone resorption drive bone loss in Eimeria infected broilers

Coccidiosis is an economically significant disease in the global poultry industry, but little is known about the mechanisms of bone defects caused by coccidiosis; thus, the study focused on effects of coccidiosis on the bone homeostasis of young broiler chickens. A total of 480 male Cobb500 broilers were randomly allocated into four treatment groups, including an uninfected control consuming diet ad libitum, two infected groups were orally gavaged with two different concentrations of sporulated Eimeria oocysts, and an uninfected pair-fed group fed the same amount of feed as the high Eimeria-infected group consumed. Growth performance and feed intake were recorded, and samples were collected on 6 days post infection. Results indicated that coccidiosis increased systemic oxidative status and elevated immune response in bone marrow, suppressing bone growth rate (P < 0.05) and increasing bone resorption (P < 0.05) which led to lower bone mineral density (P < 0.05) and mineral content (P < 0.05) under Eimeria infection. With the same amount of feed intake, the uninfected pair-fed group showed a distinguished bone formation rate and bone resorption level compared with the Eimeria infected groups. In conclusion, inflammatory immune response and oxidative stress in broilers after Eimeria infection were closely associated with altered bone homeostasis, highlighting the role of inflammation and oxidative stress in broiler bone homeostasis during coccidiosis.

Bone mineral density was reduced in the Eimeria-infected groups. Bone morphology parameters were analyzed in diaphysis and metaphysis sections using micro-computed tomography (micro-CT) ( Table 2). Tibia diaphyseal BMC was significantly reduced in the High and PF groups (P < 0.05), and diaphyseal BMD was reduced in the Low (P < 0.05), High (P < 0.05) and PF groups (P < 0.05) compared with the Control. Tibia metaphyseal BMD (total) was significantly reduced in the Low (P < 0.05) and the High (P < 0.05), whereas there www.nature.com/scientificreports/ was no significant difference between the PF group (P > 0.05) and the Control. Trabecular BMD and the number of trabecular bone (Tb. N) at metaphyseal regions were significantly reduced in the Low, High, and PF groups (P < 0.05) compared to the Control. However, metaphyseal total BMC, metaphyseal trabecular BMC, metaphyseal cortical BMD, and metaphyseal cortical BMC were not affected by Eimeria infection or feed restriction.

Eimeria-infected broilers exhibited suppression in bone formation.
The distance between double layers of calcein bands was measured at the diaphysis of the tibia and femurs to evaluate bone growth rate Table 1. Average daily feed intake (ADFI), cumulative feed intake (FI), body weight gain (BWG, kg) and feed conversion ratio (FCR) from 0 to 6 dpi. 1 Control: uninfected controls fed diet ad libitum and gavaged with water; Low: low Eimeria-infected group fed diet ad libitum and gavaged with 50,000 oocysts of E. maxima, 50,000 oocysts of E. tenella, and 250,000 oocysts of E. acervulina; High: severely Eimeria-infected group fed diet ad libitum diet and gavaged with 12,500 oocysts of E. maxima; 12,500 oocysts of E. tenella; 62,500 oocysts of E. acervulina; PF: an uninfected pair-fed group that fed the same amount of feed as the High group, gavaged with water. a,ab,b,c Treatments with different letters means a significantly difference between treatments by using Tukey's HSD test, P < 0.05, N = 10. www.nature.com/scientificreports/ ( Fig. 2; Table 3). From 0 to 4 dpi (mild infection period), the femoral growth rate was significantly decreased in the High and PF groups compared to the Control, whereas the tibial growth rate was not significantly changed during 0-4 dpi. From 4 to 8 dpi (severe infection period), tibial and femoral growth was significantly reduced in the Low group (P < 0.05) and the High group (P < 0.05), however, it was not changed in the PF group (P > 0.05) when compared with the Control. By adding data together from both injection stages, during 0-8 dpi, the Low, High, and PF treatment groups showed a significant decrease in bone formation (only femur), whereas the High group had the lowest bone growth rate (P < 0.05), followed by the Low (P < 0.05) and PF (P < 0.05) when compared with the Control. Compared with the Control, tibial growth in the Low and High groups was significantly reduced (P < 0.05) by the Eimeria challenge. However, the tibial growth of the PF group was not statistically different compared to the Control or infected groups (P > 0.05).
Relative mRNA expression of BGLAP was significantly reduced in the High group when compared with the Control (P < 0.05; Fig. 3), confirming that the severe infection of Eimeria adversely affected long bone growth. However, the expression of other bone formation markers was not significantly changed among the treatments. Moreover, higher cumulative FI was linearly correlated with higher femoral bone formation rate (R 2 = 0.4655, P = 0.010), but not in tibia (P > 0.05). The tibial bone growth rate was highly correlated with metaphyseal trabecular BMD (R 2 = 0.710, P < 0.001). Tibial bone growth rate (by calcein injection method) was positively correlated with tibial diaphysis BMD (R 2 = 0.4308, P = 0.032).
Eimeria infected broilers exhibited an increased bone resorption. The number of osteoclasts per bone surface (N. Ocl/BS) in the Low and High groups was significantly higher than in the Control (P < 0.05; Fig. 4a and b). Eimeria challenge increased the formation of TRAP-positive cells and bone resorption activity on the surface of tibia metaphysis trabecular bone. Meanwhile, an increased serum level of RANKL was observed in the Low (P < 0.05; Fig. 4c), whereas the High and PF serum levels of RANKL had numeric increasing compared to the Control (P > 0.05). Moreover, the mRNA expression of NFATC1 was significantly decreased in the PF (P < 0.05; Fig. 4d), and expression of TNFRSF11B (OPG) and TNF (tumor necrosis factor-like) was signifi- www.nature.com/scientificreports/ cantly increased in the High (P < 0.001) when compared with the Control. However, there were no significant changes in the expression of NFKB1, RANKL, FOS, ACP5(TRAP), IL1B, and SMAD1 among the treatments. A higher ratio of RANKL/OPG was observed in the Low (P < 0.05) and the PF (P < 0.05) when compared with the High (Fig. 5). Together, results suggest that Eimeria infection resulted in bone remodeling along with higher osteoclast number and activity in broilers. There was a negative correlation between TNF and metaphyseal BMD (R 2 = 0.419, P = 0.030).
Eimeria infection increased lipid peroxidation and decreased antioxidant capacity, and the correlation between redox status and bone parameters. The total antioxidant capacity of serum in the Eimeria spp. infected broilers (the Low and High groups) decreased significantly at 6 dpi compared with the non-infected Control (P < 0.05; Fig. 6a). Meanwhile, the total antioxidant capacity of serum in the PF group showed a numeric decrease compared with the Control, but the change was not statistically significant. In contrast to the antioxidant parameters in serum, the level of MDA in the liver was significantly increased by Eimeria infection compared with the Control and PF groups (P < 0.01; Fig. 6b) at 6 dpi. www.nature.com/scientificreports/ Pearson correlation analyses revealed a negative correlation of the liver TBAR level with femur growth rate (R 2 = 0.249, P = 0.005) and between the liver TBAR level and tibia growth rate (R 2 = 0.330, P = 0.001). There was a positive correlation between serum total antioxidant capacity and metaphysis bone mineral density (R 2 = 0.219, P = 0.0182); and a negative correlation between the liver TBAR level and metaphysis bone mineral density (R 2 = 0.391, P < 0.001). Meanwhile, bone marrow BGLAP mRNA level was negatively correlated with the liver TBAR level (R 2 = 0.130, P = 0.031) and positively correlated with serum total antioxidant capacity (R 2 = 0.227, P = 0.004).  www.nature.com/scientificreports/

Discussion
With increasing concerns around farm animal welfare, poultry bone abnormalities have become one of the significant challenges for the poultry industry. Bone is an essential multifunctional organ that not only provides static functions such as structural support and internal organ protection but also acts as a dynamic endocrine organ that releases hormones for mineral homeostasis, acid-base balance, and reservoir of energy and minerals 57,58 . However, for a long time, bone health in broiler production yet gained enough attention until recently because more research suggests that current commercial broiler chicken breeds grow fast to heavyweight that predisposes the chickens to leg weakness and skeletal abnormalities 59,60 . With coccidiosis so widespread, the Eimeria-challenge model was chosen as a disease model to understand the possible link between intestinal parasite infection and bone health in the current study. With a mild infection, broilers was able to compensate for growth loss partially at later stage after recover from coccidiosis, even so, the growth potential remains severely compromised 48 . Uncoupling bone remodeling may not be apparent at early growth but may show up later in the market age, resulting in clinically leg bone abnormality, eventually decreasing the market value 61 . Thus, the early stage of bone health is vital in broiler growth performance and critical in product profitability. The etiology of leg abnormalities under intestinal infection is generally complex. The factors that affect bone metabolism during infection include but are not limited to nutrition, immunity and physical stress. Eimeria infection could affect epithelium cells directly by mediating nutrition transporter activity or indirectly by causing apoptosis of cells and damaging the integrity of the intestine 3,62,63 . The damage to the digestive tract can cause malabsorption of nutrients, the deficiency of macronutrients including carbohydrates, crude protein, lipid, and minerals, and is associated with a bone remodeling imbalance that increases markers of bone resorption and decreases markers of bone formation [64][65][66] . Bone also serves as a mineral and energy reservoir, playing a role in maintaining glucose and phosphorus level within a narrow range in blood 67 . Long-term calcium deficiency is a potential risk factor for osteoporosis and bone fracture 68,69 . Depleting dietary calcium and phosphate could increase bone resorption and decrease bone mineral density 70,71 , but increased calcium intake alone is insufficient to compensate for the severe bone mineral loss under acute disease conditions 72 . Besides, protein deficiency could significantly decrease BMD and cancellous bone mass, reducing bone strength 13,14,73 . FI is positively correlated with the bone formation rate 74 . Lower energy, essential amino acid or mineral level can subsequently decrease osteoblastic activity suppressing bone formation. Moreover, nutrition deficiency could modify the plasma levels of certain essential hormones, such as growth hormone and insulin-like growth factor 75,76 , and elevated plasma stress hormones such as corticosterone [77][78][79][80][81] . Endocrine changes can directly mediate bone remodeling by regulating the activity of osteoblasts or osteoclasts, and those variables should also be considered in the experimental design. Monitoring the daily feed intake amount is very important for evaluating the growth performance especially with the impact of Eimeria infection on broiler chicken growth. In order to permit a clear interpretation of the results and limit the variables of nutrient and endocrine factors, the pair-feeding method was incorporated in this study. Pair-feeding is a technique to determine the effect of treatment on growth that is independent of nutritional factors. It has been widely used in the animal in vivo study model and was particularly well adapted to a study of energy, protein, and mineral intake deficiency 82,83 . In the current study, the amount of feed provided to the uninfected PF group was matched with the High group. However, the growth performance in the PF group was numerically higher than in the High group but not significant. The growth difference between the two groups reflected the metabolic cost of immune activation, oxidative stress, and other factors, rather than feed intake difference. Comparing the High group and the PF group provided more direct evidence to find the possible link between immune response/oxidative stress and bone metabolism. : liver lipid peroxidation was measured by TBAR assay kit, that coccidial infection significantly elevated lipid peroxidation in liver (P < 0.001). Control, non-challenge control group; Low, the low challenge dose of Eimeria; High, the high challenge dosage of Eimeria; PF, pair-feeding group that paired with High group. a,ab,b Treatments with different letters means a significantly difference between treatments by using Tukey's HSD test, P < 0.05, N = 10. www.nature.com/scientificreports/ Moreover, the micro-CT analyses revealed more specific details of bone structural changes. A significant drop in BMD was observed in bone metaphysis and diaphysis in both Eimeria-infected groups. Furthermore, the PF group and the infected Low and High groups showed different responses in the metaphysis and diaphysis sections. For example, the diaphysis is the mid-section (shaft) of a long bone and is the primary site of radial growth in young animals 84 . The diaphysis is made up of cortical bone, which has higher mineral content and density, and less water content when compared with trabecular bone, providing structural strength 85 . According to the current results, a significant decrease in BMD, BMC, and bone growth rate was observed in the diaphysis section. Both infected groups and the PF groups showed suppression of bone formation over this site. The PF group had the lowest diaphyseal BMC compared with the other groups. Lowest metaphyseal BMD was observed in the High group. It showed that bone metabolic activity appears site-specific during Eimeria infection, and bone mineral loss occurs distinctly in the proximal tibia metaphysis and diaphysis. Moreover, trabecular bones are metabolically more active than cortical bone, which not only contributes to the strength of the bone but also serves as a source of calcium for the body because trabecular bones are remodeled more rapidly during physiological processes [86][87][88] . Cortical bone loss occurs slower than trabecular loss due to the fact that less surface per unit of bone matrix volume is available for bone remodeling 89 . Nutrient deficiency, stress, and infectious skeletal disorders could cause bone mass loss and decrease bone quality by altering the trabecular bone microarchitecture 90,91 . In human and mouse research, the rate of bone turnover is more rapid in trabecular bone with a larger surface area than in cortical bone 92 . The current results showed that the bone mineral loss at the metaphysis section mainly occurred in the trabecular bone structure. Larger bone endo-surface could provide broader space for osteoblast or osteoclast attachment in trabecular bone. We hypothesized that bone resorption happens more frequently around the trabecular bone. Also, the tibial growth rate was highly correlated with tibial trabecular BMD instead of BMC, which indicates metaphysis trabecular bone BMD is more suited to evaluate early biochemical changes of bone during pathogen infection in broilers. Total mineral content (BMC) or bone ash weight may not accurately reflect the metabolic change of bone during infection.
Metabolic changes can be further assessed by different methods focusing on bone formation and resorption individually. Calcein labeling was used in the current study to visualize the newly formatted bone, and the RT-qPCR method was used to examine the bone formation-related marker genes in the bone marrow. The calcein labeling method is commonly used to assess bone growth, which directly visualizes bone growth in vivo. According to the results, by correlating FI with bone formation in each group, the Low group had significantly higher FI compared with the PF group; however, the bone formation rate was significantly lower than the PF (P < 0.05), which is in agreement with the findings of the microstructure analysis. With the same amount of FI, the PF group has a 4.71% higher BMD than the High group (P < 0.05). With a similar amount of FI (P > 0.05) between the Control and Low groups, diaphyseal BMD in the Low group was significantly lower than in the Control group. Based on the current results, even though provided with the same amount of feed, the Eimeria-challenged chickens had worse bone health status than the PF group, suggesting that apart from nutrition deficiency, other factors may be involved in bone homeostasis in both direct and indirect manners. In our previous studies, we have reported the changed redox status in broilers on 6 dpi after Eimeria spp. challenge 20 . Oxidative stress has been acknowledged as a major contributor to the immune response. The increased production of ROS is an inflammatory response that functions for the recruitment and activation of immune cells that lead to pathogen killing 93 . ROS production is involved in mineral homeostasis and contributes to bone remodeling by promoting bone resorption and suppressing bone formation. Human and mice studies have found a tight association between oxidative stress and pathogenesis of the bone disorder, that the redox state changes are related to the bone modeling and remodeling processes 94,95 . The redox state can directly impact osteoblast activity that regulates bone formation rate 96 . Oxidative stress suppressed the osteoblastic differentiation process of primary bone marrow stem cells 95 . In the current study, the oxidative stress (increased TBAR level and decreased total antioxidant capacity) was negatively correlated with bone growth rate and mRNA expression of BGLAP. This result was consistent with our previous finding 20 , that decreased bone quality was associated with systemic oxidative stress in broiler during Eimeria infection. Oxidative stress can be a co-factor involved in loss of osteoblastic activity that ultimately led to poor bone quality.
Eimeria infection can cause a complex host immune responses, encompassing both cellular and humoral mechanisms during infection 48 . Studies indicated that humoral immunity and antibodies produced by B cells were increased during severe Eimeria infection 97 . Different from other species, the bursa of Fabricius, a unique central immune organ of birds located near the cloaca, is the location of B lymphocyte differentiation and maturation instead of bone marrow 98 . Fully differentiated B lymphocytes migrate to peripheral lymphoid organs to participate in immune responses, such as producing antibodies and participating in humoral immunity 48,99 . B cell-produced proteins such as RANKL and OPG are critical for bone metabolism [100][101][102][103][104] . B lineage cells produce more than 60% of total OPG in bone marrow 102 . Mice that were injected with RANKL inhibitor resulted in a larger bone mass 105 . According to our study, an increased mRNA expression of OPG in bone marrow indicated that the system was actively producing more OPG, but the source of OPG remains unknown. The drastically higher expression of OPG in bone marrow may be related to a negative feedback loop, that increased OPG subsequently affects osteoclastogenesis 106 . We hypothesize that the bone marrow could actively reduce or inhibit highly-elevated osteoclast activity by increased expression of OPG, then preserving minerals for bone homeostasis during Eimeria infection. A similar pattern of expression was observed in gene expression of tight junction protein during acute Eimeria infection, that Eimeria infection damaged intestine integrity, but tight junction protein gene expression was significantly elevated to repair the damage 4 .
Bone marrow not only contains different cell types that perform bone formation and resorption but also serves as the cradle of hematopoiesis and a reservoir of growth factors and cytokines, providing an ideal environment for communicating between bone metabolism and the immune system. Essentially all the units that participate in cellular immunity can influence bone cells, particularly impacting the activity and formation of www.nature.com/scientificreports/ osteoclasts 2,97,107 . The cytokines IL-1β, IL-6, and TNF-α are known to increase bone resorption by stimulating both osteoclast activity and differentiation in mammals 45 . The number of osteoclast precursors increases under inflammatory conditions, characterized by high levels of the potent inflammatory cytokine TNF-α 108,109 . The current study found significantly higher mRNA expression of TNF (tumor necrosis factor-like) in the High group than in the other groups. Meanwhile, activation of osteoclast formation was detected in the High group over the metaphysis trabecular bone. It is important to mention that, with the same amount of FI, the expression of TNF was relatively low in the PF group compared with the High, as well as the number of osteoclast and enzyme activity of RANKL was relatively higher in the High group than the PF group. Based on the comparison between the High group and the PF group, we concluded that the increased osteoclastic bone resorption is associated with the activation of immune response in broiler chickens during Eimeria infection. Moreover, NFKB is involved in many signaling pathways and plays an important role in osteoclast formation and survival rate 35,110,111 . NFKB ligand (RANKL), one of the most critical molecules that regulate osteoclast formation, provides the crosstalk between bone and immune systems 32,42 . The binding of RANKL to its receptor RANK triggers osteoclast precursors to differentiate into osteoclasts, which increases the number of osteoclasts on their bone surfaces 43,112,113 . In the present study, the High group showed the lowest level of RANKL/OPG, indicating that the negative feedback loop was turned on because less osteoclastic activity was in need to preserve the minerals for bone structure and support 114 . The different expressions of the RANKL/OPG ratio between the Low and the High indicated the bone homeostasis is infection-dose-dependent during coccidiosis. However, with the difference in B-cell development in avian species, immunity, particularly humoral immunity, might interact with bone metabolism differently from mammals. How osteoimmunology plays a role in avian bone homeostasis needs more profound studies. Taken together, delayed bone development in the parasite-challenged groups was attributable to an uncoupling of osteoblast and osteoclast activity, whereby increased bone resorption and decreased bone formation were closely associated with immune response/oxidative stress during Eimeria infection. With the long-held notion that the central pathophysiology of bone disorder was nutrition deficiency and physical stress during Eimeria infection, we demonstrated that bone disorder is also closely connected with bone modeling and remodeling which are associated with immune response/oxidative stress. Both nutrition and concurrent diseases will influence the occurrence of leg disorders. Further study on osteoimmunology needs to address bone disorder issues and will further lead to a more precise understanding of the mechanism underlying the pathogenesis of bone mineral loss and bone disease in broilers, eventually improving animal production and welfare in the future.

Materials and methods
Ethics statement. The experiment followed the guideline of the Institutional Animal Care and Use Committee and was conducted at the Poultry Research Farm, University of Georgia, Athens, GA. The protocol was approved by the Institutional Animal Care and Use Committee at the University of Georgia (ethical approval code: A2021 12-012).

Experimental design.
The study was carried out in compliance with the ARRIVE guidelines. A total of 480 one-day-old male broiler chickens (Cobb 500) were randomly distributed into four treatment groups with ten replicates and twelve birds per cage. All broiler chicks were fed the same starter basal diet during day 1 to 14, and the starter diet were formulated following Cobb500 broiler management guide 115 . On day 14, all experimental groups received either water or Eimeria spp. challenge. Experimental groups included uninfected controls (Control) fed diet ad libitum (gavaged with water), a low Eimeria-infected group (Low) fed diet ad libitum (gavaged with 50,000 oocysts of E. maxima, 50,000 oocysts of E. tenella, and 250,000 oocysts of E. acervulina), a severely Eimeria-infected group (High) fed diet ad libitum diet (gavaged with 12,500 oocysts of E. maxima; 12,500 oocysts of E. tenella; 62,500 oocysts of E. acervulina), and an uninfected pair-fed group (PF; gavaged with water) that fed the same amount of feed as the High group consumed. To ensure that the pair-fed group (PF) had the same intake as the high challenge group, the amount of feed provided to each group was carefully monitored and matched. Preliminary data was used to estimate the daily feed intake of each group, and the average feed intake of the high challenge group was calculated. The same amount of feed was then provided to the pair-fed group. The feed was weighed and distributed evenly to the pair-fed group broilers at three or four intervals throughout the day (7:30 am, 3:30 pm, and 9:30 pm), with the 9:30 pm feeding time serving as an opportunity to adjust the intake of the pair-fed group to match that of the high challenge group. The grower (15-20 days of age) basal diets were formulated following Cobb500 broiler management guide 115 . Diet information is shown in Supplementary Table S1 online. All chicks were raised under the same house, feeding, and environmental management conditions based on the broiler management guide 115 . Chicks were allowed to consume water on an ad libitum basis, and daily feed intake was measured during the study. On 6 days post infection (dpi), one bird per replicate was randomly selected to collect tissue samples. The experimental design flow chart shown in Supplementary Fig. S1 online. The tissue samples were snap-frozen in liquid nitrogen and kept in − 80 °C until future processing.
Gut permeability. The gut permeability was measured on 5 dpi by the method used in our previous study 3,116 . Briefly, fluorescein isothiocyanate dextran (FITC-d; MW 4000; Sigma-Aldrich, Ontario, Canada) was dissolved in distilled water and made into 2.2 mg/mL solution. One bird per cage was randomly selected and gavaged with 1 mL of FITC-d solution. Two hours after inoculation, the blood was collected from birds and kept in the dark at room temperature for clotting. The clotted blood was centrifuged at 1500 g for 15 min to serum collection. The standard curve solution was made from a serial dilution of FITC-d stock (2.2 mg/mL). Dilution buffer was made from the pooled serum of non-infection birds with the basal diet. Sample and standard solutions were loaded into black 96-well plates, and FITC-d concentrations were measured by a spectrophotometer www.nature.com/scientificreports/ (SpectraMax M5; Molecular Devices, San Jose, CA). The excitation wavelength was set at 485 nm, and the emission wavelength was set at 528 nm.

Micro-computed tomography (micro-CT).
A total of 40 samples (one bird per cage) were randomly collected to evaluate 3-D bone morphologic changes in the broiler. The proximal and shaft of the tibia were assessed by micro-computed tomography (micro-CT). The scanning process was performed according to our previous publications 20,117 , with setting as 83 kV, 121 µA, and a 0.5 mm aluminum filter, the pixel size as 26 µm with 360° complete rotation, and 42 min of acquisition time. Scanning was performed with SkyScan 1172 (SkyScan, Kontich, Belgium). 2-D images were transferred to CTAn software (CTAn, SkyScan) for structure construction and quantification. The metaphyseal region of interest (ROI) was post-operated to automatically separate trabecular bone from cortical bone and preserve its morphology using a threshold of 800. Average bone mineral density (BMD), bone mineral content (BMC), and bone micro-architectural parameters of each group were taken from the same ROI. Cortical and trabecular bone parameters were quantified and analyzed separately. The following parameters were quantified: total volume (TV), bone volume (BV), bone surface (BS), bone volume per tissue volume (BV/ TV), and trabecular number (Tb. N) 20  Serum receptor activator of nuclear factor kappa B ligand enzyme-linked immunosorbent assay (RANKL ELISA). Chicken RANKL concentrations were measured by commercially available kits (MyBioSource, San Diego, CA, USA). All procedures were performed according to the manufacturer's protocol.
The method was two-site sandwich ELISA, the pre-coated antibody was Chicken PRM1 monoclonal antibody and the detecting antibody was polyclonal antibody with biotin labeled. A standard curve was created, and the RANKL concentration of the examined samples was calculated and expressed in pg/ml. Background OD values were subtracted from the calculation, and the color depth was directly proportional to the amount of RANKL in the sample.
Tartrate-resistant acid phosphatase staining (TRAP staining). All tibia bones were collected at 6 dpi. After removing the muscle tissue, tibias were fixed in 4% PBS-buffered formaldehyde at 4 °C for three days and then moved into 70% ethanol for preservation. Tibial tuberosity was used as a landmark to cut the bone slides by the circular saw. www.nature.com/scientificreports/ primer-blast/). The specificity of primers was validated by melting curve analysis and gel electrophoresis. RT-qPCR was performed on an Applied Biosystems StepOnePlus™ (Thermo Fisher Scientific, Waltham, MA) with iTaq™ Universal SYBR Green Supermix (BioRad, Hercules, CA) using the following conditions for all genes: 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s, annealing temperature for 20 s, and extending at 72 °C for 1 min. The geometric mean of 18S, HBMS and GAPDH were used for normalization 118 . The stability of housekeeping genes were confirmed by their consistent Ct values among the treatments (P > 0.1) 119 . BGLAP, RUNX2, SPP1, BMP2, and ALP were used as genetic markers of bone formation in the bone marrow 120 . NFKB, RANKL, FOS, ACP5, NFATC1, IL1B, TNF, SMAD1, and TNFRSF11B (OPG) 121 were used as the genetic markers for osteoclastic activity in the bone marrow. Details of primer sequences used for the experiment are presented in Table 4. Moreover, the ratio of OPG/RANKL in bone marrow was calculated. Samples were run in triplicate, and relative gene expression data were analyzed using the 2 −ΔΔCt . The mean ΔCt of each marker gene from the control group was used to calculate the ΔΔCt value, and 2 −ΔΔCt expression levels were normalized to 1 for the control group. Expression levels of the treatment groups were presented as fold change.

Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request.