A comprehensive longitudinal study quantifying the systemic effects associated with ovariectomy and high-dose corticosteroids in a sheep model of osteoporosis

Introduction

Osteoporosis is the most prevalent metabolic bone disorder in the world and the leading cause of fractures in the elderly population (1,2). A hip fracture can be detrimental to patient lifestyle and autonomy, and can even significantly increase the likelihood of mortality with fracture recurrence (3). Osteoporosis also contributes to many other orthopedic comorbidities resulting from poor bone quality. Surgical repairs of tendon and bone, such as rotator cuff tendon reattachment (4), spinal fusion (5), and limb fracture fixation (6), have a much poorer prognosis of healing in patients with osteoporotic or low-density bone. US Food and Drug Administration (FDA) recommended guidelines advise that assessment of bone tissue quality and adverse events through long-term nonclinical studies is essential in the evaluation of novel drugs intended for osteoporosis treatment (7). Therefore, reliable, robust, and well characterized animal models of low bone density are required to adequately assess the long-term efficacy and safety of medical devices and novel treatments of bone loss.

The sheep model of osteoporosis has been widely accepted as a translational large animal preclinical model to study the mechanisms of bone loss (8). These animals are particularly suitable when investigating surgical treatment of osteoporotic fractures, as they provide a comparable bone anatomy and size to humans, easily translatable for surgical hardware and medical device development (9,10). Bone loss has been studied in the sheep to evaluate several etiologies of osteoporosis, including senile (11), postmenopausal (12), and corticosteroid-induced bone loss (13). While ovariectomy (OVX) has been the primary method by which bone loss is induced in the sheep model (8), a combination of methodologies, such as administration of high-dose corticosteroids and diet manipulation in conjunction with OVX, has been shown to increase the rates of bone loss as compared to OVX alone (14-17). Additionally, corticosteroid administration in OVX sheep shows a similar phenotype to that in postmenopausal women with corticosteroid-induced osteoporosis, including decreased mineralization and bone formation rates in trabecular bone regions (18). Consequently, several studies in which sheep have received high doses of corticosteroids report potentially detrimental effects on the animal’s health due to the increased risk of infection resulting from drug-related immunosuppression (9,13,19). While bone turnover markers have been well defined in the OVX sheep model, information on the systemic impacts of model development is limited. Characterization of these parameters may further explain previously observed adverse effects of corticosteroid administration in the sheep model of osteoporosis.

Previous studies have evaluated the bone tissue and clinicopathologic parameters at long-term time points in the sheep model of osteoporosis (20-22). However, very few have directly evaluated the bone loss progression over time through serial biopsy harvest in the same animals (22,23). To our knowledge, no studies have comprehensively characterized both bone loss and clinical parameters in the sheep model via serial sampling alongside healthy control animals. Therefore, the objectives of our study were to (I) comprehensively characterize longitudinal bone loss in a 12-month sheep model of osteoporosis, and (II) report on the clinicopathologic changes throughout osteoporosis model development (i.e., administration of corticosteroids) and any potential impact on the model. We hypothesized that induction of osteoporosis in sheep would result in quantifiable bone loss, accompanied by alterations in clinical pathology parameters and steroid hormone profiles compared to age-matched controls. Furthermore, we hypothesized that acute changes in these parameters would temporally correspond with periods of corticosteroid administration. We present this article in accordance with the ARRIVE reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-2026-0112/rc).

Methods

Animals and experimental design

All procedures were approved by the Colorado State University Institutional Animal Care and Use Committee (Protocol No. 2060), in compliance with institutional guidelines for the care and use of animals, and were performed in an AAALAC-accredited facility. Sixteen healthy, skeletally mature, conventionally raised Rambouillet-cross ewes (sourced from K&S Livestock, Fort Collins, CO, USA), aged approximately 4–6 years, were enrolled in this study. Ten (N=10) ewes were randomly assigned to the osteoporosis group (OP), and six (N=6) ewes were assigned to the control group in the order they came down the chute. While allocation was randomized, no blinding of personnel occurred in this study. The proposed OP group sample size (10 sheep) was calculated using an a priori power analysis using longitudinal dual-energy X-ray absorptiometry (DXA) bone mineral density (BMD) data from a previous study (24) (G*Power version 3.1.1). This power analysis resulted in an effect size of 1.2 and a power of 90%. All animals were enrolled at the same time of year to avoid seasonal impacts of bone loss (25) and fed a standard diet of alfalfa and grass hay mix with grain supplementation, as needed. Animals were cohoused in standard indoor box pens (2.7 m × 2.7 m) for the first 2 weeks after surgical procedures, followed by turnout to dirt pasture for the remainder of the study. Osteoporosis was induced in OP sheep (N=10) via laparoscopic bilateral OVX and administration of corticosteroids, as previously described by Bisazza et al. (26). Briefly, methylprednisolone acetate (Depo-Medrol, Zoetis, Parsippany, NJ, USA) was administered starting 2 weeks following OVX for animals assigned to the OP group. Doses were administered once every 3 weeks, for a total of five doses of 500 mg intramuscularly, followed by three taper doses. Inclusion of a taper strategy was adopted to prevent significant animal side effects due to high-dose corticosteroid administration, as previously described (13). Taper doses were administered at half the previous dose at each administration (i.e., 250 mg, 125 mg, 62.5 mg). Bone imaging and ilium bone biopsy sample collection occurred at five time points for all animals: baseline (prior to OVX; denoted as “0” on graphical representation), 3 months, 6 months, 9 months, and 12 months after OVX (Figure 1). Body weights and blood collection were performed on all animals monthly throughout the study, including at baseline (Figure 1).

Figure 1 In vivo study design and sample collection time points. DXA, dual-energy X-ray absorptiometry; OP, osteoporotic sheep; OVX, ovariectomy.

DXA measurements

All animals underwent a DXA scan with a pixel size of 0.90 mm × 0.90 mm using a Hologic Discovery A scanner (Hologic, Inc., version 13.3.0.1, Marlborough, MA, USA), as previously described (26). Briefly, animals were positioned in dorsal recumbency on the DXA imaging table and scans of both the lumbar spine (L3–L5) and proximal tibia regions in the dorsoventral plane were performed. Scans were performed in triplicate and averaged together to determine a single areal BMD (aBMD) value for each site at each time point. DXA device calibration was carried out using a Hologic phantom (Hologic, Inc.) prior to each scanning time point according to the manufacturer’s protocol. aBMD was calculated using the Hologic software and reported in g/cm² for each region.

Micro-computed tomography (micro-CT) analysis

Immediately following DXA imaging at each time point, a 10 mm bone biopsy was harvested from the ilium wing using an Arthrex OATS autograft system (Arthrex, Naples, FL, USA), as previously described (26) and fixed in 10% neutral buffered formalin for a minimum of 72 hours prior to transfer to phosphate-buffered saline (PBS). The bone biopsy was used for micro-CT analysis to quantify the trabecular microarchitecture changes at the five described time points as previously described (26). Briefly, samples were scanned at a resolution of 10 µm³ at 70 kVp, 113 µA, and 500 ms integration time (Scanco µCT 80, version 1.1.15.0, Scanco USA, Inc., Wayne, PA, USA). One region of interest (ROI) (5 mm diameter, 400 slices) was drawn per sample to include only trabecular bone and reconstructed using fixed optimal threshold values (upper bound = 2,760.5 HU, lower bound = 456.7 HU). The following output measures of trabecular microarchitecture were quantified from the three-dimensional reconstruction of each ROI cylinder: bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp).

Histology and histomorphometry

Following micro-CT scanning, formalin-fixed bone biopsies were placed in a 10% ethylenediaminetetraacetic acid (EDTA) solution for one week, followed by placement in a 5% EDTA solution with solution replacement occurring weekly. Samples were then placed in xylene for three changes at 3 hours each, followed by placement into 100% reagent alcohol for 3 hours, processed in a tissue processor, paraffin-embedded, and then sectioned at 5 µm and stained with Goldner’s Trichrome. Slides were imaged at 10× magnification using an Olympus VS200 research slide scanner (Evident, Tokyo, Japan). The slide images were then semi-automatically quantified in Image-Pro Premier (version 9.3, Media Cybernetics, Rockville, MD, USA) for mean trabecular bone area (Tb.B.Ar), trabecular bone area ratio (Tb.B.Ar/Tt.Ar), mean cortical bone area (Ct.B.Ar), and mean cortical width (Ct.Wi). Mean cortical width was determined by averaging 50 perpendicular length measurements along the cortical layer of each histology sample.

Hematology and serology

Venous blood was analyzed for evaluation of hematology and serology parameters at five time points (baseline, 3, 6, 9, and 12 months). For hematology, a complete blood count (CBC) was performed on whole blood collected in EDTA tubes using an Advia 120 Hematology System (Siemens Healthineers, Munich, Germany) the same day as collection. The following hematology parameters were measured automatically by flow cytometry: hemoglobin (HGB), hematocrit (HCT), red blood cell count (RBC), mean corpuscular volume (MCV), red blood cell distribution width (RDW), mean corpuscular hemoglobin concentration (MCHC), cellular hemoglobin concentration mean (CHCM), platelets, mean platelet volume (MPV), nucleated cells, and white blood cell (WBC) differentials (lymphocytes, monocytes, eosinophils, basophils).

For serology, blood was collected in non-additive tubes, centrifuged, and the serum analyzed using a Cobas c501 analyzer (Roche Diagnostics Corporation, Indianapolis, IN, USA). The following serology parameters were automated using photometry and ion-selective electrode determinations: blood glucose, blood urea nitrogen (BUN), creatinine (CRE), phosphorus, magnesium, total protein, albumin, globulin, albumin/globulin ratio (A/G), creatine kinase (CK), total bilirubin, aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), sorbitol dehydrogenase (SDH), iron, sodium, potassium, chloride, bicarbonate, and anion gap.

Estradiol radioimmunoassay

Venous blood serum was analyzed for evaluation of estradiol levels monthly, including baseline. Estradiol (E₂) levels in sheep serum samples were measured by radioimmunoassay (RIA). One day prior to assay, 500 µL of serum sample was extracted twice by vortexing for 2 minutes with 5 mL of Diethyl Ether. The extracted serum was then frozen in a dry ice/methanol bath and the organic phase poured off and taken to dryness. Finally, 500 µL of PBS-Gel was added to the dried extract and the sample extract allowed to reconstitute overnight. The E₂ assay utilized an antibody-E₂ serum (Lot #0205041673, Eli Lilly & Co., Indianapolis, IN, USA) diluted with 1:400 normal rabbit serum in PBS-EDTA buffer (pH 7) to a working dilution of 1:400,000. The standard curve for E₂ was made by diluting purified E₂ standard to a concentration of 0.2 ng/mL in PBS-GEL (Standard 1), and serial diluting Standard 1 to create a six-point standard curve using a dilution factor of 0.4 (0.2, 0.08, 0.032, 0.0128, 0.005, and 0.002 ng/mL). The second antibody was goat anti-rabbit serum (National Wildlife Research Center, Fort Collins, CO, USA) diluted to a working dilution of 1:100 in PBS-EDTA. The ¹²⁵I-Estradiol tracer (Cat#07-138124, MP Biomedicals, Solon, OH, USA) and the quality controls were prepared with PBS-Gel: high QC 240 pg/mL, medium QC 60 pg/mL, and low QC 15 pg/mL. Sample extracts (200 µL), quality controls (80 µL), and standard (200 µL) were first incubated in 500 µL PBS-Gel buffer with 200 µL antibody and 100 µL of ¹²⁵I-Estradiol tracer, prepared with PBS-Gel, for 24 hours at 4 ℃. Then, 200 µL goat-anti-rabbit serum was added and the incubation continued for 72 hours at 4 ℃. The tubes were then centrifuged at 1,700 ×g for 30 minutes and the supernatant decanted. Finally, the radioactivity associated with each pellet was counted in a gamma counter for 1 minute. Calibration curves were determined by computer analysis using the computer program RIANAL (27). Once the calibration curve was established, it was then used to calculate the concentration of E₂ in each sample from individual runs.

Cortisol RIA

Serum cortisol values were determined by RIA according to the manufacturer’s instructions (Cortisol RIA Kit, MP Biomedicals). Briefly, serum samples and standards were added to the anti-cortisol-coated tubes. Cortisol-¹²⁵I was then added to each tube, vortexed, and incubated for 45 minutes at 37 ℃. The tubes were then counted in a gamma counter calibrated for ¹²⁵I.

Statistical analysis

All data were assessed for normality prior to statistical analysis. Differences both within (i.e., control baseline vs. control 3 months) and between groups (i.e., control vs. OP) across the study time points were determined using a mixed-effects model, and multiple comparisons were made using the Bonferroni post-hoc test. For all statistical analyses, P<0.05 was considered statistically significant (GraphPad Prism version 11.0.0; GraphPad Software, San Diego, CA, USA).

Results

Clinical results of animals

OP animal body weights were, on average, significantly lower than control animal weights at months 5 (P=0.049), 6 (P=0.008), 7 (P=0.001), 8 (P=0.027), and 12 (P=0.003). Compared to baseline values, OP body weights significantly decreased over time (1 month, P=0.02; 4 months, P=0.04; 5 months, P=0.02; 10 months, P<0.001; 11 months, P<0.001), while control body weights, on average, increased over time (6 months, P=0.03; 7 months, P<0.001; 8 months, P<0.001; 9 months, P<0.001; 10 months, P<0.001; 11 months, P<0.001; 12 months, P<0.001) (Figure S1). Nine out of 10 OP sheep were noted for significant “wool-break” and alopecia throughout the course of corticosteroid administration, with no wool loss noted in any control sheep. All wool-break had resolved in all OP animals by the study endpoint and wool growth returned to baseline presentation. One control animal was euthanized at approximately 11 months due to acute respiratory distress resulting from pulmonary caseous lymphadenitis. All other animals survived until the study endpoint of 12 months.

DXA bone density

OP L3 bone density (aBMD) was significantly lower than the control group at 3 (P=0.0005), 6 (P<0.001), 9 (P=0.004), and 12 months (P=0.043). OP L3 aBMD was also significantly lower than baseline values at 6 months (P=0.02), while no significant changes in control L3 aBMD were observed over time compared to baseline (Figure 2A). OP animals showed an increase in L3 percentage bone density loss from baseline compared to controls at 3 (−7.1%, P=0.019) and 6 months (−9.6%, P=0.010) (Figure 2B). OP L4 aBMD was significantly lower than the controls at baseline (P=0.017), 3 (P<0.001), 6 (P<0.001), 9 (P<0.001), and 12 months (P=0.006). There were no significant differences in L4 aBMD at any of the time points compared to baseline for both the OP and control groups (Figure 2C). OP animals showed an increase in percentage of L4 bone density loss from baseline compared to controls at 3 months (−7.5%, P=0.03) (Figure 2D). OP L5 aBMD was significantly lower than the control group at 3 (P=0.001), 6 (P<0.001), 9 (P=0.018), and 12 months (P=0.002). OP L5 aBMD was also significantly lower than baseline values at 6 months (P=0.006), while no significant changes in control L5 aBMD were observed over time compared to baseline (Figure 2E). The OP group showed a significant increase in percentage of L5 bone density loss from baseline compared to controls at 6 months (−9.2%, P=0.007) (Figure 2F). OP group proximal tibia aBMD was significantly lower than that of the control group at 6 (P=0.004) and 9 months (P=0.02). OP group proximal tibia aBMD was significantly increased at 3 months (P=0.004), but then significantly decreased at 6 (P=0.01) and 9 months (P=0.041) compared to baseline values. Control values were greater at 3 (P=0.004), 6 (P=0.042), and 12 months (P=0.041) compared to baseline (Figure 2G). The OP group showed a significant increase in percentage of tibia bone density loss from baseline compared to controls at 6 (−6.6%, P<0.001) and 9 months (−5.6%, P=0.003) (Figure 2H).

Figure 2 Dual-energy X-ray absorptiometry aBMD at baseline, 3, 6, 9, and 12 months. (A) Lumbar vertebra L3 aBMD and (B) % change in L3 aBMD from baseline (Time 0). (C) Lumbar vertebra L4 aBMD and (D) % change in L4 aBMD from baseline. (E) Lumbar vertebra L5 aBMD and (F) % change in L5 aBMD from baseline. (G) Proximal tibia aBMD and (H) % change in tibia aBMD from baseline. Raw aBMD measurements are represented as box and whisker plots with individual animals marked. % change in aBMD is represented as mean ± SD. For all panels, sample numbers for baseline, 3, 6, and 9 months are N=6 control, N=10 OP; for 12 months: N=5 control, N=10 OP. Comparisons made using a mixed-effects model and Bonferroni post-hoc comparison of means. Within-group comparisons indicated by solid significance line; between-group comparisons indicated by dashed significance line. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. aBMD, areal bone mineral density; OP, osteoporotic sheep; SD, standard deviation.

Ilium wing micro-CT trabecular microarchitecture

OP group ilium wing biopsy bone volume fraction was significantly lower at 3 (P=0.049) and 9 months (P=0.003) than control group values. OP group BV/TV was significantly lower at 9 months (P=0.02) compared to baseline values. No significant changes from baseline in BV/TV were observed in the control group across time points (Figure 3A). OP group bone biopsy Tb.N was significantly lower than control group values at 6 months (P=0.037). No significant differences were observed within control and OP groups across time points (Figure 3B). OP group bone biopsy Tb.Sp was significantly greater than control group values at 6 months (P=0.005). OP group Tb.Sp was also significantly greater than baseline values at 6 months (P=0.028). No significant differences in control Tb.Sp values were noted across time points (Figure 3C). OP group bone biopsy Tb.Th was significantly lower at 9 months than control group values (P=0.003). OP group Tb.Th was also significantly lower at 9 months (P=0.023) compared to baseline values. All other time point and group comparisons were not significantly different (Figure 3D).

Figure 3 Micro-computed tomography bone microarchitecture of iliac crest bone biopsies at baseline, 3, 6, 9, and 12 months. (A) BV/TV, (B) Tb.N, (C) Tb.Sp, and (D) Tb.Th. Data are represented as box and whisker plots with individual animals marked. For all panels, sample numbers for baseline, 3, 6, and 9 months are N=6 control, N=10 OP; for 12 months: N=5 control, N=10 OP. Comparisons made using a mixed-effects model and Bonferroni post-hoc comparison of means. Within-group comparisons indicated by solid significance line; between-group comparisons indicated by dashed significance line. *P<0.05, **P<0.01. BV/TV, bone volume fraction; MicroCT, micro-computed tomography; OP, osteoporotic sheep; Tb.N, trabecular number; Tb.Sp, trabecular spacing; Tb.Th, trabecular thickness.

Bone histomorphometry

Qualitative assessment of ilium wing bone biopsy histology slides revealed progressive thinning and disconnection of trabecular bone in OP sheep over time (Figure 4A-4E), while minimal disruptions to trabecular connectivity and thickness are shown in control sheep bone throughout the study (Figure 4F-4J). No differences were observed in control histomorphometry parameters at any time point compared to baseline values, and no differences were observed between OP and control groups at baseline (Figure 5A-5D). No significant differences in histomorphometry parameters were noted between or within groups at 3 months. At 6 months, OP bone Tb.B.Ar was significantly lower than control values (P=0.035), as well as significantly decreased as compared to baseline values (P=0.017) (Figure 5A). At 9 months, OP group bone Tb.B.Ar (P<0.001) and Tb.B.Ar/Tt.Ar (P=0.006) decreased significantly when compared to control values at the same time point (Figure 5A,5B). No significant differences in trabecular histomorphometry parameters (Tb.B.Ar, Tb.B.Ar/Tt.Ar) were noted between or within groups at 12 months (Figure 5A,5B), and no significant differences in cortical histomorphometry parameters (Ct.B.Ar, Ct.Wi) were noted between or within groups at any time point (Figure 5C,5D). One 9-month OP biopsy sample was excluded from cortical bone histomorphometry analysis, as no cortical bone was present in the histology sample.

Figure 4 Representative histology images of iliac crest bone biopsies from single animal in the OP group at (A) baseline, (B) 3 months, (C) 6 months, (D) 9 months, and (E) 12 months. (F-J) Representative images of bone biopsies from single animal in the control group at (F) baseline, (G) 3 months, (H) 6 months, (I) 9 months, and (J) 12 months. Goldner’s Trichrome staining; scale bar: 2 mm. OP, osteoporotic sheep.

Figure 5 Histomorphometry outcomes for bone biopsies. (A) Tb.B.Ar, (B) Tb.B.Ar/Tt.Ar, (C) Ct.B.Ar, and (D) Ct.Wi. Data are represented as box and whisker plots with individual animals marked. For all panels, sample numbers for baseline, 3, 6 months: N=6 control, N=10 OP; for 9 months: N=6 control, N=9 OP; for 12 months: N=5 control, N=10 OP. Comparisons made using a mixed-effects model with the Bonferroni post-hoc comparison of means. Within-group comparisons indicated by solid significance line; between-group comparisons indicated by dashed significance line. *P<0.05, **P<0.01, ***P<0.001. Ct.B.Ar, cortical bone area; Ct.Wi, cortical width; OP, osteoporotic sheep; Tb.B.Ar, trabecular bone area; Tt.Ar, total area.

Hematology and serology analysis

No differences in whole blood hematology values were observed between OP and control groups at baseline. At 3 months, OP group platelet counts (P<0.001), MPV (P=0.007), and MCV (P=0.039) were significantly greater than control animals (Table 1, Figure S2). Significant differences in WBC percentages were also noted at 3 months between groups, with increased neutrophil percentage observed in the OP group (P=0.009), as well as decreased monocytes (P=0.01) and lymphocytes (P=0.007) when compared to the control group (Table 1, Figure S2). At 6 months, eosinophils (P=0.046) and RBC counts were lower in OP animals (P=0.001), along with an increase in MCV (P<0.001) (Table 1, Figure S2). No hematological differences were observed at 9 and 12 months between groups.

BUN values differed between OP and control groups at baseline (P=0.009). No other serological values differed between groups at baseline. At 3 months, serum phosphorus (P<0.001), GGT (P<0.001), sodium (P<0.001), chloride (P=0.006), bicarbonate (P<0.001), and potassium (P=0.02) levels were elevated in OP animals compared to control animals. Serum magnesium (P=0.02), total protein (P<0.001), albumin (P=0.021), BUN (P<0.001), CRE (P=0.001), and bilirubin (P=0.02) were all statistically decreased in OP animals compared to controls at 3 months (Table 2, Figure S3). At 6 months, OP animals showed significant increases in serum iron (P<0.001) and sustained increases in phosphorus (P=0.004) and GGT (P<0.001) levels. OP BUN (P<0.001), CRE (P=0.006), total protein (P=0.007), and albumin (P=0.03) levels also remained lower than controls at 6 months (Table 2, Figure S3). At 9 months, CK (P=0.03) levels were lower in OP animals compared to controls. At 12 months, OP group animals showed lower total protein (P=0.046) levels compared to controls (Table 2, Figure S3). While additional differences in hematological and serological parameters were noted within groups between time points as compared to baseline (Figures S2,S3), we focus primarily on the comparisons between OP and control group samples at each time point to account for natural fluctuations in sheep blood clinical pathology parameters.

Steroid hormone analysis

Serum cortisol was significantly lower in OP animals than controls at 3 months (P<0.001) and higher than control values at 9 (P<0.001) and 10 months (P=0.004) post-OVX (Figure 6A). OP serum cortisol values were notably closer to zero than those of control animals between 1 and 5 months post-OVX, consistent with the corticosteroid administration time frame. Fasting appeared to potentially have an impact on cortisol levels, resulting in an increase in cortisol levels at fasting time points (baseline, 3, 6, and 9 months) for both groups. To account for this effect, fasted time points were then also compared separately from unfasted time points for cortisol (Figure 6B). Estradiol levels of OP animals were significantly lower than those of control animals at months 1 (P=0.042), 4 (P=0.009), 8 (P<0.001), and 10 (P=0.001) post-OVX (Figure 6C). Fasting also appeared to potentially impact estradiol levels, resulting in a decrease in estradiol concentrations at fasting time points for both groups (Figure 6D). OP group animals showed both decreased fasted cortisol and estradiol levels at 3 months (P=0.005, P=0.008, respectively) compared to controls, as well as increased fasted cortisol levels at 9 months (P=0.001) compared to controls (Figure 6B,6D).

Figure 6 Steroid hormone levels in blood serum. (A) Serum cortisol levels of all study animals at monthly intervals. (B) Serum cortisol levels at fasted time points only (baseline, 3, 6, and 9 months). (C) Serum estradiol levels of all study animals at monthly intervals. (D) Serum estradiol levels at fasted time points only. Data are represented as mean ± SD. Individual animals are marked on (B) and (D) only. For all panels, sample numbers for baseline to 11 months: N=6 control, N=10 OP; for 12 months: N=5 control, N=10 OP. Comparisons made using a mixed-effects model with the Bonferroni post-hoc comparison of means. Within-group comparisons indicated by solid significance line; between-group comparisons indicated by dashed significance line. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. OP, osteoporotic sheep; SD, standard deviation.

Discussion

The sheep has been a useful large animal model to study osteoporosis and perform long-term preclinical studies on bone quality. Induction of osteoporotic bone loss in sheep via both OVX and corticosteroid administration significantly decreases the bone density and disrupts trabecular microarchitecture over a relatively short period of time (15-17,26,28). As expected, we observed decreases in lumbar vertebrae and tibial bone density compared to controls, with more drastic changes in vertebral bone density due to a higher ratio of trabecular bone at the lumbar spine. This was observed in conjunction with comparable changes in bone biopsy microarchitecture by 6 months, including decreasing bone volume fraction, trabecular number, and trabecular thickness and increasing trabecular spacing compared to controls over time. Histomorphometrically, we observed significant decreases in trabecular bone area in our OP animals compared to baseline values at 3 and 6 months, with no changes observed in our control group between time points. We also qualitatively observed changes to the trabecular bone quality in our OP animals compared to the controls, such as thinning and disconnection of the trabeculae following osteoporotic induction. This trend in histological trabecular bone loss of our osteoporotic group is in alignment with previous sheep studies (28,29), where the most significant bone loss manifested by 3–6 months following OVX and corticosteroid administration. Additionally, no difference in cortical bone area or width was observed between groups. Given that trabecular bone loss is the primary source of bone loss in early osteoporosis (30-32), it is expected that significant changes would occur to the trabecular bone and minimal changes to the cortical surfaces during our study duration.

While most sheep osteoporosis studies focus primarily on the bony changes in these animals, few have explored clinicopathological changes in the same model, including hormone levels and hematological parameters. Coelho et al. reported hematology and biochemistry parameters at baseline and at 6 months in the sheep osteoporosis model; however, no control group was reported for comparison (28). Since clinical pathology reference ranges for conventionally raised sheep are underreported and present high variability, we aimed to evaluate changes in the systemic parameters of osteoporotic sheep over time alongside a control group. Additionally, previous studies have rarely noted side effects in the animals due to high-dose corticosteroid administration (13,33) but have not explored the clinical impact of model development in these animals in detail. Given that osteoporosis is a complex metabolic condition, it is important that the holistic changes occurring in our animal models of osteoporosis are accounted for—not just the impact on the bone. In this study, we compared systemic values of steroid hormones, hematological, and serological parameters in an ovine model of osteoporosis over a time frame of 12 months.

In the present study, we note the greatest number of significant differences in hematological parameters between control and OP group animals at 3 and 6 months following OVX. This included significant impacts on WBC differentials, platelet parameters, and RBC values. We attribute many of these changes to be most likely related to the administration of corticosteroids, as administration of the final high dose of corticosteroids was concluded at 15 weeks (approximately 3 months), with the final taper dose administered at 24 weeks (approximately 6 months). The minimal differences we observed in clinicopathological values after 6 months could subsequently be attributed to a medication recovery period. However, since we did not allocate a group of animals to receive OVX only on this study, it remains difficult to ascertain with certainty which of the hematological and serological effects were due to corticosteroid administration versus OVX. Notably, the increase in neutrophils and subsequent decrease in lymphocytes, monocytes, and eosinophils is consistent with what has been historically reported in the human clinical literature for patients undergoing treatment with high-dose corticosteroids (34-36), where neutrophilia is commonly accompanied by a decrease in monocytes and eosinophils. However, some outcomes in our study were not as comparable to the human literature, including anemia and increased serum iron levels noted at 6 months. These outcomes may be more specific to sheep and require further investigation. The hematological findings from our osteoporotic group animals align with values reported previously by Coelho et al. (28), where comparisons were made between sheep at baseline and 6 months following osteoporosis induction. We note similar hematological parameter changes at 6 months, including decreased RBC counts with increased MCV, as well as comparable trends with our 3-month data. While it was important that we compare values within the animals over time, our study highlights differences between a control group undergoing the same biopsy procedures and anesthesia events. Clinical blood values are wide ranging in sheep, making it difficult to ascertain meaning of results without a control group.

Previous studies have also reported alopecia, or “wool-break”, in sheep which were administered high-dose or long-acting corticosteroids (13,33,37). This phenomenon was a notable side effect also observed in most of our study animals, with nine out of ten osteoporotic group animals displaying some form of significant wool shedding by 6 months. At 3 months, serum cortisol levels were significantly diminished in the osteoporotic group as compared to control animals, but returned to normal levels following the cessation of corticosteroid administration. Similar trends in endogenous serum cortisol have also been reported previously in osteoporotic sheep (33), suggesting that adrenal insufficiency may be induced in these animals early in the model development, requiring the implementation of taper doses to prevent widespread infection resulting from immunosuppression. Given the timing of alopecia and corresponding low endogenous cortisol levels in our osteoporotic group, we can potentially infer that there is a correlation between the two events. High levels of stress and endogenous cortisol have been linked to “wool-break” and decreased wool fiber length in sheep (38-40). Endogenous cortisol is synthesized in the adrenal cortex and is under the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Under normal physiologic conditions, corticotropin-releasing hormone (CRH) is released from the hypothalamus, which in turn stimulates the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary. ACTH acts on the adrenal cortex to synthesize and release cortisol. Endogenous cortisol then inhibits the secretion of CRH and ACTH through a negative feedback loop mechanism (41). Supraphysiologic doses of corticosteroids can also act to inhibit the HPA axis by binding to glucocorticoid receptors, preventing the release of CRH and ACTH, and ultimately suppressing the synthesis and release of endogenous cortisol (42). Since administration of high-dose corticosteroids can flood the body with synthetic cortisol, one possible explanation for the observation of alopecia in our osteoporotic animals may be that the corticosteroids are generating a physiologic high-stress state for the body and resulting in loss of wool.

Removal of the ovarian production of estradiol has been shown to significantly decrease systemic levels of estradiol in rats following bilateral OVX, a common small animal model of osteoporosis (43,44), mimicking trends of estradiol in postmenopausal women (45). In contrast, in the sheep model described here, we noted a significant decrease in serum estradiol levels in our osteoporotic group animals at select months (1, 3, 4, 8, and 10 months) following ovary removal compared to controls, with levels continuing to fluctuate closely with the control group. This observed bounce-back of serum estradiol, although differing from humans and rats, is consistent with previous sheep osteoporosis studies (15,23,46). While the ovaries are the primary source of endogenous 17β-estradiol, the most potent form of estrogen in mammals, estradiol is also produced in multiple other extragonadal organs such as the adrenal glands, skin, and adipose tissue (47). In humans, adipose tissue is considered to be the major secondary source of circulating estrogen, as estradiol plays a beneficial role in lipogenesis (48). Although not investigated in this current study, based on the literature evidence regarding human sources of extragonadal estrogen, we hypothesize that these extragonadal sources of estradiol synthesis may be a source of estrogen compensation for the loss of the ovaries in our ovariectomized sheep and contribute to minimal changes in circulating levels of estradiol (23). However, to our knowledge, estrogen levels have not been directly investigated in non-gonadal tissue in any ruminant species. Additionally, it is well established that alfalfa hay is a source of phytoestrogens (49,50), including genistein (isoflavone), apigenin (flavone), and coumestrol (coumestan), which have been demonstrated to have estrogenic effects by acting as a ligand for estrogen receptors (51-53). Phytoestrogenic pastures have also been linked to negative impacts on sheep and cattle fertility (54,55). While there is little to no evidence in the literature demonstrating that these phytoestrogens composing alfalfa have a direct impact on circulating estradiol levels in sheep, phytoestrogen binding to estrogen receptors may offer a weak protective mechanism for bone in sheep, as is observed in humans (56). This potential binding of phytoestrogens to estrogen receptors could also contribute to the low percentage bone loss that is observed when OVX alone has historically been used to model osteoporosis in sheep (23). However, the alfalfa feed utilized in this study was not analyzed for phytoestrogen content and therefore its effects on sheep estradiol levels and on bone density are unknown. Further investigation into extragonadal and external sources of estradiol synthesis in sheep is required to understand the impact on circulating estradiol levels and, thus, on bone homeostasis following OVX.

It is crucial to clearly define the differences in osteoporosis clinical presentation between our animal models and the human pathologies those models aim to represent, as this information is essential in the model selection process. To our knowledge, our study was the first to comprehensively evaluate both the bone and systemic effects of osteoporosis induction in a sheep model by serial biological sample collection alongside seasonal and age-matched controls over one year. Unlike small animal models, sheep provide a robust model in which long-term studies can be conducted with a wide array of sample collections of multiple tissue types at timed intervals.

We acknowledge that this study is not without its limitations. Primarily, the use of conventionally raised cross-bred sheep provides additional complexities to the study population. There is high variability in ranges for conventionally raised sheep clinical pathology values, but we attempted to control for this variable by enrollment of a control group. Additionally, there may have been other underlying medical conditions within animals that could have impacted the results. For example, the animal that was euthanized early due to a case of caseous lymphadenitis did not present until immediately prior to euthanasia. While all other animals did not clinically present with any interfering pathologies prior to or during the study, it is possible that other animals had underlying medical conditions which could have impacted the interpretation of clinical pathology results. As these are conventionally raised animals, subject age was also limited to visualization of eruption of incisors (57). We made every effort to narrow the age of these animals by excluding animals younger than 4 years and ewes with broken or significantly worn-down teeth during the study selection process. Since age has been reported to impact bone density in sheep (11), it is possible that the subtle differences in age between animals could have also impacted the hematology, serology, and potentially the estradiol levels, as well. Another limitation of the study is that we did not perform endpoint organ histopathology and therefore could not assess any damage to organs that may have been incurred due to corticosteroid administration over the duration of model development (i.e., kidney, liver, adrenal gland). Sheep were not screened for bone density prior to enrollment onto the study; therefore, there were some disparities between groups at baseline; notably, the L4 bone density and the BUN values showed significant differences between OP and control animals at baseline. To account for bone density differences observed between groups at baseline and minimize bias of interpretation, we reported the percentage change of bone loss at each BMD assessment site alongside raw BMD value differences. Due to inherent differences in bone density between our study animals at baseline, percentage change in bone density is a more accurate assessment when comparing between groups. Additionally, any differences in BUN noted at baseline between groups, while statistically different, are clinically within normal limits and should not affect interpretation of group differences at any other time points. No other differences were noted between groups at baseline. Lastly, not all blood collection time points were fasted, making trends in steroid hormones difficult to discern longitudinally across groups. Additionally, stressors such as handling and venipuncture can impact cortisol levels. However, animals were handled in a similar fashion and at the same time intervals across groups.

Conclusions

In conclusion, we report on systemic parameters in an established sheep model of osteoporosis via OVX and corticosteroid administration. We demonstrate the effect of model development on steroid hormone levels, hematology, and serology in the same animals over the course of 12 months via serial sampling. Comprehensive characterization of the clinical manifestations of the sheep model is necessary for its appropriately use in preclinical studies of osteoporosis.

Acknowledgments

The authors would like to thank Terry Nett and team at the Endocrinology Laboratory at Colorado State University for performing the steroid hormone assays; the Clinical Pathology Laboratory at the Colorado State University Veterinary Teaching Hospital for performing the CBC and diagnostic chemistry blood panels; and Liz Chlipala at Premier Laboratories, LLC for histology slide preparation. The authors would also like to thank Dr. Eric Gilleland for statistical consultation. Special acknowledgment to the Translational Medicine Institute anesthesia team for performing anesthetic procedures, and the Preclinical Surgical Research Laboratory animal care staff for the highest quality care of the animals throughout the study.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-2026-0112/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-2026-0112/dss

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-2026-0112/prf

Funding: This study was funded by the Preclinical Surgical Research Laboratory at Colorado State University.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-2026-0112/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Animal experiments were performed under an approved project protocol (No. 2060) granted by the Colorado State University Institutional Animal Care and Use Committee, in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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