Source: TEXAS A&M UNIVERSITY submitted to NRP
BISPHOSPHONATE PHARMACOKINETICS AND COMPREHENSIVE EFFECTS ON JUVENILE CARTILAGE, BONE GROWTH AND HEALING: IMPLICATIONS FOR ANIMAL WELFARE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1025068
Grant No.
2021-67015-34079
Cumulative Award Amt.
$500,000.00
Proposal No.
2020-05281
Multistate No.
(N/A)
Project Start Date
Jul 1, 2021
Project End Date
Jun 30, 2025
Grant Year
2021
Program Code
[A1251]- Animal Health and Production and Animal Products: Animal Well-Being
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Animal Science
Non Technical Summary
Widespread "off-label" use of bisphosphonates to alter bone development in young horses is common in the equine industry, however the effects of these drugs have not been tested in horses of this age. This practice may contribute to the early breakdown of young horses, which, beyond being a major concern for animal welfare, may also put the health and safety of jockeys in danger. The goal of the work is to determine the effects of bisphosphonate use on bone and joint health in young horses undergoing exercise through the use of a horse and sheep model. Specifically, to determine the clearance of bisphosphonate from the bloodstream in young horses/sheep, determine the time course of influence and effects of bisphosphonates on bone development, and determine the effects of bisphosphonates on cartilage when subjected to an inflammatory insult. We expect that younger animals will clear bisphosphonates from their system faster than adult horses, that bone development will be compromised leading to easier breakdown, and that bisphosphonates will have anti-inflammatory effects in the joint. This research will create knowledge to drive future research regarding the use of bisphosphonates in young horses and will provide evidence upon which new regulatory policies for the use of bisphosphonates in young horses will be based.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3153810102075%
3153610102025%
Knowledge Area
315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3610 - Sheep, live animal; 3810 - Horses, ponies, and mules;

Field Of Science
1020 - Physiology;
Goals / Objectives
Themajor goalsof this proposal are to determine the wide-ranging effects of bisphosphonate administration in healthy, skeletally immature horses with specific regard to horses undergoing sales preparation and early exercise training. Bisphosphonates are known to have contrasting effects: 1) inhibition of bone resorption with the potential to cause bone microdamage and 2) anti-inflammatory, analgesic, and potential chondroprotective effects in the joint. Through the proposed study, investigators will assemble a comprehensive dataset evaluating the various effects of bisphosphonates in juvenile animals in order to aid in education and regulation of bisphosphonate administration.Thespecific aimsinclude:Aim 1:To determine acute dose pharmacokinetics of clodronate in juvenile ovids and equids.Aim 2:To determine the relative time course and overarching effects of bisphosphonate administration on bone formation and remodeling under forced exercise through the use of a dual model approach using serum biomarkers of bone modeling/remodeling, endocrine markers of bone regulation, microstructure analyses, and biomechanical testing.Aim 3:To determine intra-articular effects of bisphosphonates in juvenile, exercising horses through the use of a minimally invasive model of acute synovitis developed in our laboratory.
Project Methods
Pharmacokinetics:The pharmacokinetic study will utilize 20 juvenile animals (n = 10 horses; n = 10 sheep) assigned to receive a bisphosphonate administration on day 0 of each trail. Body weight (BW) will be recorded, and an initial blood sample will be harvested prior to intramuscular administration of clodronate at the recommended dose of 1.8 mg/kg BW in horses and 0.6 mg/kg BW in sheep. Blood samples will be collected 0.5, 1, 3, 6, 12, 24, 48, and 72 hours post-administration and centrifuged for plasma collection. Samples from both the sheep and horse will be stored at -80°C and shipped frozen to a commercial laboratory for bisphosphonate quantification by LC-MS. Data will be used to determine maximum concentration (Cmax), area under the curve plasma concentrations (AUC), plasma half-life (T1/2), and time of maximum concentration (Tmax).Experiment I:Yearling horses (n=32) will be used for a 168-day experimental period separated into two phases. Phase I: days 0-84 for sales preparation; Phase II: days 85-168 for exercise training. Horses will be housed in individual stalls with the exception of designated exercise. Diets will be formulated to meet or exceed nutrient requirements for yearling horses (NRC, 2007).Horses will be stratified, and treatments will be balanced by age, BW, sex, and initial radiographic bone aluminum equivalence (RBAE) into one of four treatment groups consisting of a control group (CON; n = 8), a single dose group (1X; n=8) receiving a dose at the start of Phase II, a two-dose group (2X; n = 8) receiving doses at the start of Phase I and Phase II, and a four-dose group (4X; n=8) receiving doses at the start of and halfway through each phase. Clodronate will be administered intramuscularly at 1.8 mg/kg BW. Investigators will be blinded to treatments, which will be color coded by the pharmacy.In Phase I, horses will exercise at a walk and trot 5 days per week beginning at 25 minutes and increasing 5 minutes per week until a total of 45 minutes is reached and continued until the end of Phase I. In Phase II, all groups will be exercised 5 days per week, 30 minutes per day with a program including walk, trot, and canter that will increase in intensity throughout Phase II. Exercise will be performed through the use of a free-stall exerciser.Beginning on day 0, every 42 days: BW, body condition score (BCS), lameness evaluation, and morphometric measurements will be recorded. Lameness evaluations will be determined by an evaluator using a 0-5 scale outlined by the American Association of Equine Practitioners. Blood samples will be collected via jugular venipuncture into non-additive blood tubes to harvest serum. Blood samples will be centrifuged and stored at -80°C until further analysis. Radiographic imaging will be used to determine the presence of sesamoiditis and bone mineral density using RBAE.Serum samples harvested throughout the study will be analyzed for various biomarkers of bone formation and resorption to quantify bone metabolism. Biomarkers will be analyzed using commercially available ELISAs and include: Procollagen I N-terminal propeptide (PINP), bone-specific alkaline phosphatase (BALP), tartrate-resistant acid phosphatase isoform 5b (TRAP 5b), carboxy telopeptide of type I collagen crosslaps (CTX-1), and receptor activator of nuclear factor NF-κB ligand (RANKL). Biomarkers to assess endocrine regulation of bone development include serum Ca2+concentrations, growth hormone (GH) and parathyroid hormone (PTH).On day 84, a biopsy of the left tuber coxae will be performed, again on day 168, a similar biopsy will be harvested from the ipsilateral and contralateral tuber coxae. Samples will be analyzed by microCT to assess changed in bone structure and potential damage.On day 126, all groups will undergo an intra-articular lipopolysaccharide (LPS) challenge.Briefly, radial carpal joints will be randomly assigned to receive either 0.5 ng sterileEscherichia coliO55:B5 derived LPS or an equivalent volume of sterile lactated Ringer's solution as a contralateral control. Synovial fluid samples will be collected via sterile arthrocentesis prior to LPS injection (0 hour) and at 6, 12, 24, and 336-hour post-injection and stored at -80°C. Samples will be analyzed for prostaglandin E2 (PGE2), interleukin 6 (IL-6), carboxypeptide of type II collagen (CPII) and catabolic collagenase cleavage neopeptide (C2C) using commercially-available ELISAs.Data collected will be analyzed using the MIXED procedure of SAS v9.4. Non-normal data will be transformed. Horse will serve as a random effect with main effects of treatment, time, and treatment x time interaction.Experiment II:Dorset-Polypay sheep (n=40), approximately 80% of mature size, will be subjected to exercise and clodronate treatment to compliment the equine study over a period of 168 days. Animals will be housed with access to outdoor paddocks and fed a dry total mixed ration daily to meet dietary requirements.Sheep will be stratified by sex and age and randomly assigned to one of four treatment groups: control (CON; n = 10), T0(n = 10) receiving clodronate at day 0, T0+84(n = 10) receiving clodronate at day 0 and 84, and T84(n = 10) receiving clodronate at day 84. Clodronate will be administered intramuscularly at 0.6 mg/kg.Exercise will be performed through the use of a free stall exerciser 4 days per week, increasing in duration beginning at 10 minutes with 5 minute increases each week until 30 minutes is reached and maintained to the end of the project. Once maximum duration has been reached, intensity will be increased.Every 14 days, a complete physical examination will be performed including heart rate, respiratory rate, body temperature, BW and height. A lameness examination will be performed using a subjective 0-6 scale. Beginning on day 0 and every 28 days, blood will be collected via jugular venipuncture into non-additive blood collection tubes and allowed to clot. Blood will be centrifuged and stored at -80°C until later analysis.Serum samples will be analyzed for the same bone biomarkers analyzed in the equine study. Additionally, C-telopeptide fragments of type II collagen (CTX-II) will be measured using a commercially available ELISA.Bone biopsies of the tuber coxae will be obtained on day 84 and following euthanasia on day 168 similarly to the equine study.On day 168, animals will be humanely euthanized following final blood collection. Select major organs will be harvested, weighed immediately, and a biorepository of frozen and fixed tissue samples will be stored for future analysis. For the present study, the fused metacarpus, lumbar spine, tuber coxae, and mandible will be utilized. The fused metacarpus and lumbar vertebrae will undergo strength testing using 4-point bending and axial compression tests to determine bone resistance to fracture. The lumbar spine, mandible and tuber coxae will be utilized forMicroCT analysis.Data collected will be analyzed using a repeated measures ANOVA evaluating the main effects of treatment, time, and treatment x time interaction. Data will be transformed if non-normal.Effortswill include experiential learning opportunities for undergraduate students that assist with either the animal orlab work phases of the research projects. Further, publication and presentation of findings through both scientific journals and meetings, as well as extension programs to extend knowledge to industry professionals.Evaluationwill include:Establishment of an advisory group that represents scientists, veterinarians, principal stakeholders, and industry professionals to objectively assess and evaluate the annual progress toward measurable outcomes and potential impacts.Preparation of abstracts, manuscripts, and popular press articles, as appropriate.Contribution of knowledge to equine regulatory agencies to promote the welfare and well-being of horses, regardless of breed or discipline.

Progress 07/01/21 to 06/30/25

Outputs
Target Audience:During the final year of the project, scientists and industry professionals have been reached by presentation of posters at scientific conferences, publication of a dissertation, publication of interviews in popular press media, and publication of peer-reviewed journal articles. Specifically: 1) two abstracts and posters were presented at the Equine Sciences Society Conference in Ft. Collins, CO in June 2025. 2) Brittany Paris graduated in December 2024after defending her dissertation in August 2024. 3)three interviews have been published for lay audiences (two in 2025 and 1 in 2021 that was not previously reported) a)Ledbetter, K. 2021. Texas A&M Study to Evaluate Bisphosphonate Risk in Young Horses (popular press article about a portion of the project). https://agrilifetoday.tamu.edu/2021/05/05/texas-am-study-to-evaluate-bisphosphonate-risk-in-young-horses/ b)Pfeffer, H. 2025. Effects of Bisphosphonates on Yearling Horse Joints (popular press article about a portion of the project). https://thehorse.com/1137695/effects-of-bisphosphonates-on-yearling-horse-joints/ c) Pfeffer, H. 2025. Clodronate Disodium's Effects on Bone Density and Structure in Young Horses (popular press article about a portion of the project)https://thehorse.com/1137649/clodronate-disodium's-effects-on-bone-density-and-structure-in-young-horses/ 4) Details about the threejournal articles are presented in the Products portion of this report, Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the final year of the project, onestudent (Brittany Paris)received a Doctor of Philosophy degree in Animal Science from Texas A&M University in December 2024. Over the course of the project two students earned M.S. degrees (from Texas A&M University) and two students earned Ph.D. degrees (one from Texas A&M University and one from Michigan State University), Previous annual progress reports contain the students' names and titles of their theses and dissertations. Multiple undergraduate students acquiredhigh impact experiential learning experience over the course of this project at Texas A&M University, Michigan State University, and Montana State University (previous annual progress reports contained more specific examples of the project's impact on training and education of undergraduate students. How have the results been disseminated to communities of interest?Dissemination of the results to communities of interest (scientists, equine veterinarians, and horse industry members) was accomplished bypresentations (oral and poster formats)at a scientific conference, participation ininterviews for a major industry publication (The Horse), and publication of scientific articles. Specifically: 1) two presentations at the Equine Sciences Society Conference in Ft. Collins, CO in June 2025 (detailed in Products portion of this report). 2)three interviews for lay audiences appeared in 2025 (2 articles) and 2021 (1 article that was not previously reported) a)Ledbetter, K. 2021.Texas A&M Study to Evaluate Bisphosphonate Risk in Young Horses(popular press article about a portion of the project). https://agrilifetoday.tamu.edu/2021/05/05/texas-am-study-to-evaluate-bisphosphonate-risk-in-young-horses/ b)Pfeffer, H. 2025.Effects of Bisphosphonates on Yearling Horse Joints(popular press article about a portion of the project). https://thehorse.com/1137695/effects-of-bisphosphonates-on-yearling-horse-joints/ c) Pfeffer, H. 2025. Clodronate Disodium's Effects on Bone Density and Structure in Young Horses(popular press article about a portion of the project)https://thehorse.com/1137649/clodronate-disodium's-effects-on-bone-density-and-structure-in-young-horses/ 3) The details about three of the project's most recent journal articles are presented in the Products portion of this report, What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Clodronate disodium (CD) is a bisphosphonate, a class of bone modulating drugs. It has been used extra-label in juvenile horses, despite only being approved for horses greater than 4 years of age. As juvenile, exercising horses undergo high levels of bone turnover, there is concern regarding the effects of extra-label use of bisphosphonates, such as CD, due to lack of scientific data to inform this extra-label use. This project was conducted with an ovine model and an equine model: 1) to estimate the plasma pharmacokinetics of CD; 2) determine the effects of CD on endocrine regulation of bone; 3) determine bone turnover using circulating levels of biomarkers that reflect bone formation and bone resorption; 3) to assess CD's effect on circulating levels of biomarkers that reflect bone formation and bone resorption; and 4) to evaluate the effects of CD on biomarkers of cartilage metabolism and intra-articular inflammation within the articulating joint of the juvenile horse using an intra-articular inflammatory model. Liquid chromatography-tandem mass spectrometry analysis determined that CD is detectable in equine plasma up to 72 h post-administration (mean maximum concentration was attained at 0.5 h post-administration; mean apparent elimination half-life was 28.9 h). The half-life estimated for CD in the current study of juvenile Quarter Horses was different than previously published data in mature horses. All physical growth measurements increased over time but were not affected by treatment. Concentrations of parathyroid hormone increased following a second bisphosphonate dose on d 84 and 126 for the 4-dose and 2-dose treatment groups, respectively. Growth hormone concentrations tended to decrease over time while ionized calcium concentrations decreased over time. When undergoing low intensity exercise, horses that received CD had lower serum concentrations of a biomarker of osteoclast number and activity, reflecting a decrease in the cells responsible for bone resorption. There were no differences in other biomarkers of bone resorption or bone formation. In the ovine model, we examined the effects of CD on bone turnover, serum bone biomarkers (SBB), bone mineral density (BMD), bone microstructure, biomechanical testing (BT), and cartilage glycosaminoglycan content (GAG) over 165 days. CD did not have any measurable effects on the skeleton of sheep. SBB showed changes over time, with increases in bone formation and decreases in some bone resorption markers. Tuber coxae biopsies showed increasing bone volume fraction, trabecular spacing and thickness, and reduced trabecular number on day 165 versus day 84. These changes may be attributed to exercise or growth. The absence of treatment effect may be explained by the lower CD dose used in large animals compared to humans. While clodronate disodium reduced serum TRAP5b, indicative of osteoclast number and activity, when administered during a low intensity exercise program, it did not impact other systemic biomarkers of bone resorption or formation in juvenile horses. When administered extra-label in juvenile horses at the dose approved by the FDA for horses 4 years of age and older, CD may not be sufficient to alter systemic measures of bone metabolism. Further research is needed to examine whether low doses of bisphosphonates may be used in active juvenile populations for analgesia without evidence of bone changes. Information is needed regarding the local effects of clodronate disodium administration on weight bearing bones. This initial investigation into the effects of CD on the juvenile articulating joint, lays the groundwork for further investigations into the effects of CD administration in the juvenile synovial joint.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Conrad, M.B., J.L. Leatherwood, B.L. Paris, J.M. George, R.E. Martinez, F.B. Vergara-Hernandez, B.D. Nielsen, A.C. Colbath, C.E. Arnold, K.G. Glass, T.H Welsh Jr., and A.N. Bradbery. 2025. Effects of clodronate disodium on endocrine regulators of calcium in yearling horses. Journal of Animal Science. 103:skaf132, https://doi.org/10.1093/jas/skaf132.
  • Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2025 Citation: George, J.M., J.L. Leatherwood, J.L. Leatherwood, B.L. Paris, C.E. Arnold, K.G. Glass, M.B. Conrad, R.E. Martinez, F.B. Vergara-Hernandez, B.D. Nielsen, A.C. Colbath, T.H. Welsh, Jr., and A.N. Bradbery. 2025. Effects of clodronate disodium on markers of inflammation and cartilage metabolism in juvenile horses challenged with intra-articular lipopolysaccharide. Journal of Animal Science (accepted for publication on September 26, 2025).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: George, J.M., J.L. Leatherwood, A.N. Bradbery, C.E. Arnold, K.G. Glass, B.L. Paris, M.B. Conrad, R.E. Martinez, A.S. Reiter, C.R. Kerth, and T.H. Welsh, Jr. 2025. Effects of bisphosphonate administration on synovial metabolome of juvenile horses challenged with intra-articular lipopolysaccharide. Journal of Equine Veterinary Science, 148:105511. https://doi.org/10.1016/j.jevs.2025.105511.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Paris, B.L., J.L. Leatherwood, J.M. Popovich, K.G. Glass, C.E. Arnold, N.E. Bray, M.B. Conrad, J.M. George, R.E. Martinez, F.B. Vergara-Hernandez, A.C. Colbath, B.D. Nielsen, T.H. Welsh, and A.N. Bradbery. 2025. Clodronate disodium did not impact tuber coxae microarchitecture or bone mineral density in juvenile horses. Journal of Equine Veterinary Science, Volume 148:105515. https://doi.org/10.1016/j.jevs.2025.105515.
  • Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Paris, B.L. 2024. Bisphosphonate Pharmacokinetics and Comprehensive Effects on Juvenile Bone Growth and Healing. Ph.D. Dissertation. Texas A&M University, College Station, TX. https://oaktrust.library.tamu.edu/items/c0df89d1-9655-4c72-9e1d-2953d1a3b024 (https://hdl.handle.net/1969.1/1593814)
  • Type: Peer Reviewed Journal Articles Status: Submitted Year Published: 2026 Citation: Paris, B.L., J.L. Leatherwood, T.H. Welsh, Jr., C.E. Arnold, K.P. Glass, M.B. Conrad, J.M. George, R.E. Martinez, A.C. Colbath, B.D. Nielsen, and A.N. Bradbery. Clodronate disodium reduced TRAP5b independent of affecting other systemic biomarkers of bone resorption or formation in juvenile, exercising Quarter Horses. Journal of Animal Science (Submitted).


Progress 07/01/23 to 06/30/24

Outputs
Target Audience:During the third study period, scientists and industry professionals have been reached by presentation of posters at scientific conferences. A group of industry members was presented with some information from the project at the American Quarter Horse Association Convention- Racing Committee Meeting on March 17, 2024. This presentation was coordinated with help of a member of the stakeholder committee. Changes/Problems:Experiment 1:There were no significant changes or problems associated with experiment 1 during the second study period. Experiment 2:There were no significant changes or problems associated with experiment 2 during the second study period. What opportunities for training and professional development has the project provided?Four graduate students were trained in proper research methods, sample analyses, statistical analyses, and scientific writing. Graduate students working on Experiments 1 & 2 travelled to one scientific conference during the third study period to present research and further develop expertise in their areas of study related to the project. One abstract (Conrad et al., 2024) presented was a result of additional funding secured from the American Quarter Horse Foundation to analyze samples collected for the biorepository associated with the current study. One undergraduate student was trained in sample analyses, statistical analyses, scientific writing, and presentation. Completion of the remaining objectives will facilitate the remaining PhD candidate earning advanced degree in animal sciences. How have the results been disseminated to communities of interest?Experiment 1:Presentation of RBAE and lameness data via poster at scientific conference (July 2024). The poster was awarded first place in the PhD II Student Poster Competition. Presentation of microCT methods by an undergraduate student; the poster received first place in Health-Sciences at the Michigan State University Undergraduate Research & Arts Forum. Experiment 2:Publications of 2 manuscripts in open access journals describing the use of juvenile sheep as an exercising model for large animals and the effects of clodronate disodium in the juvenile, exercising ovine model. What do you plan to do during the next reporting period to accomplish the goals?Completion of analyses of equine radiographs for sesamoiditis. Publication of equine pharmacokinetics manuscript. Publication of equine endocrine markers of bone regulation manuscript. Publication of equine serum biomarkers of bone turnover manuscript. Publication of equine intra-articular lipopolysaccharide challenge manuscript. Publication of equine radiograph, microCT, and lameness manuscript. Further dissemination of information to scientific and lay target audiences.

Impacts
What was accomplished under these goals? Experiment 1: Aim 1 Clodronate pharmacokinetics were re-analyzed using a commercially available pharmacokinetic software. Manuscript edits are underway. Aim 2During the third study period, radiograph analysis for radiographic bone aluminum equivalence (RBAE) was completed. There were no treatment effects on RBAE. Further, analyses for data collected from the bone biopsy microCT scans were completed. There were no differences in measures of microarchitecture or bone mineral density based on treatment administrations. Radiographs collected for sesamoiditis are being analyzed by a licensed Large Animal Veterinary Surgeon. Preparation is underway for three manuscripts: endocrine markers of bone development; biomarkers of bone modeling/remodeling; RBAE, sesamoiditis, microCT, and lameness. Aim 3Manuscript writing is underway for the markers of cartilage metabolism and inflammation. Experiment 2: Two manuscripts were published during the second reporting period. The second publication from experiment 2 evaluates a novel exercise model in sheep. This study evaluated adaptation to the exercise protocol, physical and lameness examinations every 15 days, and serum bone biomarkers of bone formation and bone resorption analysis every 28 days in control sheep. This manuscript has been published. The third manuscript from experiment 2 reports the effects of clodronate disodium in the juvenile, exercising ovine model. The manuscript includes analysis of serum bone biomarkers, biomechanical testing, CT analysis of bone density, microstructural analysis (Micro-CT), and cartilage assessment. No treatment effects were detected. This manuscript has been published.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Vergara-Hern�ndez, F.B., B.D. Nielsen, C.L Panek, C.I. Robison, and A.C. Colbath. (2023). Exercising sheep as a preclinical model for musculoskeletal research. Am. J. Vet. Res. 85(3):1-8. doi:10.2460/ajvr.23.09.0209
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Vergara-Hern�ndez, F.B., B.D. Nielsen, J.M. Popovich, C.L. Panek, A.A. Logan, C.I. Robison, R.A. Ehrhardt, T.N. Johnson, N.J. Chargo, T.H. Welsh, Jr., A.N. Bradbery, J.L. Leatherwood, and A.C. Colbath. (2024). Clodronate disodium does not produce measurable effects on bone metabolism in an exercising, juvenile, large animal model. PLoS ONE. 19(4):e0300360. doi:10.1371/journal.pone.0300360
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Vergara-Hernandez, F. B. Nielsen, J. Popovich Jr., and A. Colbath. (2023). #41 - Investigating the impact of clodronate disodium on bone metabolism using a juvenile, exercising sheep model for juvenile horses. 17th World Equine Veterinary Congress Proceedings (abstract). Santiago, Metropolitan Region, CL. November, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Bray, N.E., B.L. Paris, F.B. Vergara-Hernandez, A.M. Bradberry, K.P. Glass, C.I. Robison, R.A. Ehrhardt, T.H. Welsh Jr., J.L. Leatherwood, A.C. Colbath, B.D. Nielsen, and J.M. Popovich, Jr. (2024). Assessment of location and size dependent bone microarchitecture differences in equine tuber coxae. University Undergraduate Research and Arts Forum (abstract), Michigan State University, East Lansing, Michigan, USA. April 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Paris, B.L., J.L. Leatherwood, T.H. Welsh, Jr., K.G. Glass, C.E. Arnold, M.B. Conrad, J.M. George, R.E. Martinez, A.C. Colbath, B.D. Nielsen, and A.N. Bradbery. (2024). PSVI-12 Clodronate does not impact bone optical density or lameness in juvenile, exercised Quarter Horses. American Society of Animal Science Annual Meeting Proceedings (abstract), Calgary, Alberta, CA. July, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Conrad, M.B., J.L. Leatherwood, K.G. Glass, C.E. Arnold, B.L. Paris, J.M. George, R.E. Martinez, T.P. Mays, and A.N. Bradbery. (2024). PSV-4 Clodronate re-release in response to controlled exercise and bone stressors in juvenile horses. American Society of Animal Science Annual Meeting Proceedings (abstract), Calgary, Alberta, CA. July, 2024.
  • Type: Theses/Dissertations Status: Published Year Published: 2023 Citation: Vergara Hernandez, F.B. 2023. The effects of clodronate disodium on equine joint tissues and osseous metabolism: From in vitro analysis to a pre-clinical, ovine model under exercise. PhD diss. Michigan State Univ., East Lansing, Michingan. https://www.proquest.com/docview/2877293743?pq-origsite=gscholar&fromopenview=true&sourcetype=Dissertations%20&%20Theses


Progress 07/01/22 to 06/30/23

Outputs
Target Audience:During this reporting period, scientists and industry professionals have been reached by presentation of posters at scientific conferences. The stakeholder group was presented with data that has been generated from the project up to this point and plans for future work were discussed through a stakeholder meeting which was held on August 21, 2023, via Zoom. Changes/Problems:Experiment 1: There were no significant changes or problems associated with experiment 1 during the second study period. Experiment 2:One serum bone biomarker (PINP) was excluded from analysis for one day due to lack of samples. No other significant problems or changes were encountered during the study. What opportunities for training and professional development has the project provided?Four graduate students were trained in proper research methods, sample analyses, statistical analyses, and scientific writing. Graduate students working on Experiment 1 travelled to two scientific conferences during the second study period to present research and further develop expertise in their areas of study related to the project. Completion of the remaining objectives will facilitate PhD candidates earning advanced degrees in animal sciences. How have the results been disseminated to communities of interest?Experiment 1: Presentation of serum endocrine biomarkers of bone regulation, serum biomarkers of bone turnover, and synovial biomarkers of cartilage metabolism via oral presentation at scientific conference (June 2023). Presentation of juvenile equine clodronate pharmacokinetics and synovial biomarker of inflammation via poster presentation at scientific conference (July 2023). Experiment 2: Presentation of abstract at scientific conference (Oct 2022). Publication in open access manuscript describing the pharmacokinetics of juvenile sheep. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, the following will be accomplished: Completion of analyses of equine radiographs and microCT images. Publication of equine pharmacokinetics manuscript. Publication of equine endocrine markers of bone regulation manuscript. Publication of equine serum biomarkers of bone turnover manuscript. Publication of equine intra-articular lipopolysaccharide challenge manuscript. Publication of juvenile, exercising ovine model manuscript. Publication of clodronate on juvenile, exercising ovine. Further dissemination of information to scientific and lay target audiences.

Impacts
What was accomplished under these goals? Experiment 1: Aim 1 Clodronate in yearling horses exhibited bicompartmental kinetics compared to linear in mature horses, which will inform manufacturers, veterinarians, and researchers of differential drug kinetics in skeletally immature horses. Maximum clodronate concentrations were much higher in yearling horses dosed at the same rate as mature horses (1.8 mg/kg BW). During the second study period, pharmacokinetic data was submitted for presentation at the American Society of Animal Science 2023 Annual Meeting. An abstract was published in the conference proceedings and the poster was presented on July 18, 2023. The poster presentation received 2nd place in the PhD II Poster Competition. Manuscript preparation is underway with plans to submit in the early portion of the third study period. Aim 2 During the second study period, sample analyses for biomarkers of bone modeling/remodeling and endocrine markers of bone development were completed. There were no effects of clodronate on growth hormone, calcitonin, or ionized calcium. There was a treatment by time interaction on parathyroid hormone (PTH), where PTH increased following the second clodronate injection in both the two and four dose groups. An abstract for the endocrine biomarkers of bone turnover was submitted in February 2023 for the 2023 Equine Science Society Meeting and was published in the conference proceedings and presented on June 7, 2023, in Grapevine, TX. Manuscript preparation is currently underway. There were no effects of clodronate on receptor activator of nuclear factor kB ligand (RANKL), c-terminal crosslinks of type I collagen (CTX-1), bone-specific alkaline phosphatase (BAP), or procollagen type I n-terminal propeptide (PINP). There was a treatment by time interaction on tartrate resistant acid phosphatase 5b (TRAP5b), where TRAP5b decreased to day 126 in the two and four dose groups, while remaining the same or increasing in control and the one dose group. An abstract on the biomarkers of bone metabolism was submitted in February 2023 for the 2023 Equine Science Society Meeting and was published in the meeting proceedings and presented on June 7, 2023, in Grapevine, TX. Manuscript preparation is currently underway. Radiographs collected for radiographic bone aluminum equivalence (RBAE) are currently undergoing analysis and will be completed during the third study period. Radiographs collected for sesamoiditis are being analyzed by a licensed Large Animal Veterinary Surgeon and will be completed during the third study period. Scanning of bone biopsy samples for microstructure through MicroCT has been completed by Michigan State University. Data from the resulting scans are undergoing analyses and will be completed during the third study period. Aim 3 Analyses of markers of cartilage metabolism and inflammation were completed during the second study period. Clodronate treatments indicate a tendency to increase cartilage synthesis in response to intra-articular lipopolysaccharide injection, but there was no effect of treatment on cartilage degradation. Inflammatory biomarkers indicated a three-way interaction for clodronate treatment groups, indicating clodronate increases intra-articular inflammation following an insult via intra-articular lipopolysaccharide and does not decrease inflammation compared to a control. An abstract for the cartilage biomarkers was submitted in February 2023 for the 2023 Equine Science Society Meeting and was published in the meeting proceedings and presented on June 7, 2023, in Grapevine, TX. An abstract on synovial inflammation was submitted for the American Society of Animal Science Annual Meeting and was published in the meeting proceedings and presented as a poster on July 18, 2023, in Albuquerque, NM. Manuscript writing is currently underway. Experiment 2: Data analyses included serum biomarkers, CT scans (bone density), mechanical testing, micro-CT scans (microstructural measurements and bone density), and analysis of sulfated glycosaminoglycan content of articular cartilage. Three publications were anticipated from this work: The first publication describes the pharmacokinetics of clodronate disodium in juvenile sheep. The dose chosen (0.6 mg/kg) from preliminary studies corresponded remarkably well to the published pharmacokinetics in horses. This study has been published in the American Journal of Veterinary Research. The second publication evaluates a novel exercise model in sheep. This study evaluated adaptation to the exercise protocol, physical and lameness examinations every 15 days, and serum bone biomarkers of bone formation and bone resorption analysis every 28 days in control sheep. This manuscript is ready for submission. The third manuscript is in progress. This manuscript reports the effects of clodronate disodium in the juvenile, exercising ovine model. The manuscript includes analysis of serum bone biomarkers, biomechanical testing, CT analysis of bone density, microstructural analysis (Micro-CT), and cartilage assessment. No treatment effects were detected.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Vergara-Hernandez, F.B., Nielsen, B.D., and Colbath, A.C. (2022). Is the use of bisphosphonates putting horses at risk? An osteoclast perspective. Animals. 12(13):1722. doi:10.3390/ani12131722
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Vergara-Hernanadez, F.B., Nielsen, B.D., Kottwitz, J.J., Panek, C.L., Robison, C.I., Paris, B.L., Welsh, T.H., Bradbery, A.N., Leatherwood, J.L., and Colbath, A.C. (2023). Pharmacokinetics and plasma protein binding of a single dose of clodronate disodium are similar for juvenile sheep and horses. Am. J. Vet. Res. 84(8) doi:10.2450/ajvr.23.03.0051
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Conrad, M.B., Leatherwood, J.L., Paris, B.L., George, J.M., Martinez, R.E., Vergara-Hernandez, F.B., Nielsen, B.D., Colbath, A.C., Arnold, C.E., Glass, K.G., Welsh, T., and Bradbery, A.N. (2023). 2 Effects of extra-label bisphosphonate use on growth parameters and endocrine markers of bone health in juvenile horses. J. Equine Vet. Sci. 124:104304. (abstract) Equine Science Society Symposium, Grapevine, Texas. June, 2023. doi:10.1016/j.jevs.2023.104304
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: George, J.M., Leatherwood, J.L., Arnold, C.E., Glass, K.G., Paris, B.L., Conrad, M.B., Martinez, R.E., Vergara-Hernandez, F.B., Nielsen, B.D., Colbath, A.C., Welsh, T.H., and Bradbery, A.N. (2023). 11 Extra-label bisphosphonate effects on cartilage turnover in juvenile horses challenged with intra-articular lipopolysaccharide. J. Equine Vet. Sci. 124:104313. (abstract) Equine Science Society Symposium, Grapevine, Texas. June, 2023. doi:10.1016/j.jevs.2023.10431
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Paris, B.L., Leatherwood, J.L., Arnold, C.E., Glass, K.G., Conrad, M.B., George, J.M., Martinez, R.E., Vergara-Hernandez, F.B., Colbath, A.C., Nielsen, B.D., Welsh, T.H., and Bradbery, A.N. (2023). 10 Extra-label bisphosphonate effects on bone turnover biomarkers in juvenile, exercising horses. J. Equine Vet. Sci. 124:104312. (abstract) Equine Science Society Symposium, Grapevine, Texas. June, 2023. doi:10.1016/j.jevs.2023.104312
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: George, J.M., Leatherwood, J.L., Arnold, C.E., Glass, K.G., Paris, B.L., Conrad, M.B., Martinez, R.E., Vergara-Hernandez, F.B., Nielsen, B.D., Colbath, A.C., Welsh, T.H., and Bradbery, A.N. (2023). PSIII-8 Extra-label bisphosphonate effects on intra-articular inflammation in juvenile horses challenged with intra-articular lipopolysaccharide. American Society of Animal Science Annual Meeting Proceedings (abstract), Albuquerque, New Mexico. July, 2023. DOI not yet assigned.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Paris, B.L., Leatherwood, J.L., Welsh, T.H., Arnold, C.E., Glass, K.G., Conrad, M.B., George, J.M., Martinez, R.E., Linne, P.L., Mays, T.P., Colbath, A.C., Nielsen, B.D., and Bradbery, A.N. (2023). PSVI-8 Bisphosphonate pharmacokinetics in juvenile horses. American Society of Animal Science Annual Meeting Proceedings (abstract), Albuquerque, New Mexico. July, 2023. DOI not yet assigned.


Progress 07/01/21 to 06/30/22

Outputs
Target Audience:During this reporting period, scientists and industry professionals have been reached by presentation of posters at scientific conferences. Efforts include atotal of 26 undergraduate students were trained and taught proper research methods to earn a total of 52 research credit hours across two semesters. Changes/Problems:Experiment 1: One animal had to be removed from the study due to an unrelated respiratory infection, which required anti-inflammatory medication to manage fever and prevented the horse from receiving a treatment injection on day 126. Another animal was euthanized due to a fracture of the tibia during the exercise protocol on day 163. Prolonged healing of horses following ipsilateral and contralateral bone biopsies on day 168 of the project caused delays in beginning the sample analyses for aims 2 and 3. Additionally, backorder of assays due to pandemic supply chain issues has delayed progress for Aim 2 biomarkers of bone modeling/remodeling and endocrine markers of bone development. Assay validation and sample analysis remains underway. No other problems regarding sample analysis have been encountered. Experiment 2: Minimal problems were encountered during the study period. One animal had to be removed from exercising the second half of the study due to lameness associated with the tuber coxae biopsies. No other significant problems or changes were encountered during the study. What opportunities for training and professional development has the project provided?Undergraduate students learned to properly and safely handle young horses, observed collection of blood and tissue samples, assisted in sample processing, and gained knowledge on writing scientific research papers. Five graduate students were trained in proper research methods, sample collection, and sample analyses. Completion of the remaining objectives will facilitate graduate students earning advanced degrees in animal sciences. How have the results been disseminated to communities of interest?Experiment 1: Presentation of growth data via poster at scientific conference. A popular press article regarding how the current research will be used to promote health of the horse was released to the public. https://agrilifetoday.tamu.edu/2021/05/05/texas-am-study-to-evaluate-bisphosphonate-risk-in-young-horses/ Experiment 2: Presentation of poster at a scientific conference. What do you plan to do during the next reporting period to accomplish the goals?Experiment 1: Aim 1 Data will be submitted for presentation at relevant scientific conferences. Manuscript preparation is underway. Aim 2 Sample analysis for biomarkers of bone modeling/remodeling and endocrine markers of bone development began in April 2022 and remains ongoing. Radiographs collected for radiographic bone aluminum equivalence (RBAE) are currently undergoing analysis. Samples for analysis of microstructure have been delivered to Michigan State University for MicroCT, and analysis is ongoing. Aim 3 Markers of synovial inflammation are currently being analyzed. During the second study period, analyses for Aim 2, bone and endocrine markers, will be completed and undergo statistical analysis. Analyses for Aim 3, intra-articular health, will be completed and undergo statistical analysis. Abstracts will be submitted for presentation at the June 2023 meeting of the Equine Science Society in Grapevine, TX. Manuscript preparation of equine data will also commence during the second study period. Experiment 2: During the second study period, bone biomarker data will be analyzed. In addition, microCT analysis will be completed for tuber coxae biopsies, whole tuber coxae (to assess past biopsy sites) and a small portion of MC3/4. Three publications are anticipated to be completed during the second study period. The first will focus on the pharmacokinetics of clodronate disodium in juvenile sheep. The second will describe the sheep exercise model. This work will help future researchers seeking to use sheep for musculoskeletal disease studies. The third will report the effects of clodronate disodium in the juvenile, exercising ovine model.

Impacts
What was accomplished under these goals? Experiment 1: Aim 1 Sample collection for Aim 1 was completed June 2021, sample analysis was completed by the collaborators at Texas Veterinary Medical Diagnostic Laboratory (TVMDL), and pharmacokinetic data analysis was completed by Montana State University in February 2022. Clodronate in yearling horses exhibited bicompartmental kinetics compared to linear in mature horses, which will inform manufacturers, veterinarians, and researchers of differential drug kinetics in skeletally immature horses. Aim 2 Sample collection for the equine study Aim 2 was completed in December 2021. Physical data, including body weight, body condition score, wither height, hip height, body length, and heart girth circumference underwent statistical analysis in January 2022. No treatment effects were detected. An abstract was submitted in March for the 2022 American Society of Animal Science-Canadian Society of Animal Science Annual Meeting and was presented as a poster on June 29, 2022, in Oklahoma City, OK. Aim 3 Sample collection for Aim 3 of the equine study was completed in November 2021. To date, analysis of both markers of cartilage metabolism have been completed. Experiment 2: The goal for the portion of the project completed at Michigan State University during the study period was to assess the effects of clodronate on a juvenile, exercising, sheep model. During the reporting period, 39 of 40 sheep completed the 24-week exercise protocol. One sheep had lameness following the tuber coxae biopsy and exercise was discontinued. All samples were obtained as scheduled including blood and tuber coxae biopsies pre-mortem as well as all post-mortem samples. We completed a pharmacokinetic study which will provide information for future researchers seeking to utilize sheep as a model for clodronate administration in horses. The dose chosen (0.6 mg/kg) from preliminary studies corresponded remarkably well to the published pharmacokinetics in horses. Manuscript preparation is under way for these results. In addition, serum biomarkers of bone formation, bone resorption and cartilage degradation were collected throughout the study (every 4 weeks). Final data analysis is underway for serum biomarkers. Biomechanical testing of the fused MC3/4 and a lumbar spinal vertebrae was completed during the project period. No treatment effects were found. Likewise, CT analysis of bone density was completed for lumbar spinal, mandible, and fused MC3/4. No treatment effects were found.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Conrad, M. B., J. L. Leatherwood, K. G. Glass, C. E. Arnold, B. L. Silvers, J. M. George, R. E. Martinez, B. D. Nielsen, A. C. Colbath, T. H. Welsh, Jr., A. N. Bradbery. 2022. Clodronate use does not influence physical growth parameters in yearling horses undergoing forced exercise. ASAS-CSAS Annual Meeting Proceedings. Oklahoma City, OK. Poster No. PSXV-14.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Vergara-Hernandez, F. B., B. D. Nielsen, C. L. Panek, C. I. Robison, J. J. Kottwitz, J. M. Jr. Popovich, A. A. Logan, R. A. Ehrhardt, and A. C. Colbath. 2022. Effects of clodronate disodium: a juvenile exercising sheep model. Sixth An. Musculoskelet. Heal. Symp. Poster no. 43. doi:10.13140/RG.2.2.26969.39525.