Clodronate disodium does not produce measurable effects on bone metabolism in an exercising, juvenile, large animal model.

Bisphosphonates are commonly used to treat and prevent bone loss, but their effects in active, juvenile populations are unknown. This study examined the effects of intramuscular clodronate disodium (CLO) on bone turnover, serum bone biomarkers (SBB), bone mineral density (BMD), bone microstructure, biomechanical testing (BT), and cartilage glycosaminoglycan content (GAG) over 165 days. Forty juvenile sheep (253 ± 6 days of age) were divided into four groups: Control (saline), T0 (0.6 mg/kg CLO on day 0), T84 (0.6 mg/kg CLO on day 84), and T0+84 (0.6 mg/kg CLO on days 0 and 84). Sheep were exercised 4 days/week and underwent physical and lameness examinations every 14 days. Blood samples were collected for SBB every 28 days. Microstructure and BMD were calculated from tuber coxae (TC) biopsies (days 84 and 165) and bone healing was assessed by examining the prior biopsy site. BT and GAG were evaluated postmortem. Data, except lameness data, were analyzed using a mixed-effects model; lameness data were analyzed as ordinal data using a cumulative logistic model. CLO did not have any measurable effects on the skeleton of sheep. SBB showed changes over time (p ≤ 0.03), with increases in bone formation and decreases in some bone resorption markers. TC biopsies showed increasing bone volume fraction, trabecular spacing and thickness, and reduced trabecular number on day 165 versus day 84 (p ≤ 0.04). These changes may be attributed to exercise or growth. The absence of a treatment effect may be explained by the lower CLO dose used in large animals compared to humans. 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.

Evaluation of dietary arginine supplementation to increase placental nutrient transporters in aged mares.

Nine pregnant mares (18.2 ±â€…0.7 yr; 493.82 ±â€…12.74 kg body weight [BW]) were used to test the hypothesis that dietary supplementation of l-arginine would enhance placental vascularity and nutrient transport throughout gestation in aged mares. Mares were balanced by age, BW, and stallion pairing, and assigned randomly to dietary treatments of either supplemental l-arginine (50 mg/kg BW; n = 7) or l-alanine (100 mg/kg BW; n = 6; isonitrogenous control). Mares were individually fed concentrate top-dressed with the respective amino acid treatment plus ad libitum access to Coastal Bermudagrass hay. Treatments began on day 14 of gestation and were terminated at parturition. Mare BW, body condition score (BCS), and rump fat were determined, and body fat percentage was calculated every 28 d and concentrate adjusted accordingly. Doppler blood flow measurements including resistance index (RI) and pulsatility index for uterine artery ipsilateral to the pregnant uterine horn were obtained beginning on day 21 and continued every 7 d until day 154 of gestation, and prior to parturition. Parturition was attended with foaling variables and placental measures recorded. Placental tissue from the pregnant horn was analyzed histologically to assess cell-specific localization of vascular endothelial growth factor (VEGF) and cationic amino acid transporter 1 (SLC7A1) proteins. Semiquantitative analyses were performed using 10 nonoverlapping images per sample fixed in a 10× field (Fiji ImageJ v1.2). Mare performance data were analyzed using PROC MIXED in SAS and foaling and placental data were analyzed using PROC GLM. Gestation length at parturition was not influenced (P > 0.05) by supplemental arginine. Compared with arginine-supplemented mares, control mares had a thicker rump fat layer (P < 0.01) and greater percent body fat (P = 0.03), and BCS (P < 0.01) at parturition. Arginine-supplemented mares had a lower RI than control mares prior to parturition (P < 0.01). Body length, height, and BW of foals at birth, as well as placental weight and volume, and immunohistochemical staining for VEGF and SLC7A1 at parturition, were not affected (P > 0.05) by maternal arginine supplementation. These results indicate that dietary arginine supplementation (50 mg/kg BW) is safe for gestating mares. A larger number of mares is required to extend knowledge of effects of supplemental arginine on embryonic/fetal survival and growth in mares.

Pharmacokinetics and plasma protein binding of a single dose of clodronate disodium are similar for juvenile sheep and horses.

OBJECTIVE: To determine the single-dose pharmacokinetics of clodronate disodium (CLO) in juvenile sheep and the plasma protein binding (PPB) of CLO in juvenile sheep and horses. ANIMALS: 11 juvenile crossbred sheep (252 ± 6 days) for the pharmacokinetic study. Three juvenile crossbred sheep (281 ± 4 days) and 3 juvenile Quarter Horses (599 ± 25 days) for PPB analysis. METHODS: CLO concentrations were determined using liquid chromatography-mass spectrometry. Pharmacokinetic parameters were calculated by noncompartmental analysis from plasma samples obtained at 0, 0.5, 1, 3, 6, 12, 24, 48, and 72 hours after CLO administered IM at 0.6 mg/kg. PPB was determined using equine and ovine plasma in a single-use rapid equilibrium dialysis system. RESULTS: The mean and range for maximum plasma concentration (Cmax: 5,596; 2,396-8,613 ng/mL), time of maximal concentration (Tmax: 0.5; 0.5-1.0 h), and area under the curve (AUCall: 12,831; 7,590-17,593 h X ng/mL) were similar to those previously reported in horses. PPB in sheep and horses was moderate to high, with unbound fractions of 26.1 ± 5.1% in sheep and 18.7 ± 7.5% in horses, showing less than a 1.4-fold difference. CLINICAL RELEVANCE: The pharmacokinetic parameters and PPB of CLO in juvenile sheep were similar to those previously reported in horses. The results suggest that juvenile sheep can be utilized as an animal model for studying the potential risks and/or benefits of bisphosphonate use in juvenile horses.

Equine enterocytes actively oxidize l-glutamine, but do not synthesize l-citrulline or l-arginine from l-glutamine or l-proline in vitro.

In livestock species, the enterocytes of the small intestine are responsible for the synthesis of citrulline and arginine from glutamine and proline. At present, little is known about de novo synthesis of citrulline and arginine in horses. To test the hypothesis that horses of different age groups can utilize glutamine and proline for the de novo synthesis of citrulline and arginine, jejunal enterocytes from 19 horses of three different age groups: neonates (n = 4; 7.54 ± 2.36 d of age), adults (n = 9; 6.4 ± 0.35 yr), and aged (n = 6; 22.9 ± 1.0 yr) with healthy gastrointestinal tracts were used in the present study. Enterocytes were isolated from the jejunum and incubated at 37 °C for 30 min in oxygenated (95% O2/5% CO2) Krebs bicarbonate buffer (pH 7.4) containing 5 mM D-glucose and 0 mM, 2-mM L-[U-14C]glutamine, or 2 mM L-[U-14C]proline plus 2 mM L-glutamine. Concentrations of arginine, citrulline, and ornithine in cells plus medium were determined using high-performance liquid chromatography. Results indicate that the rate of oxidation of glutamine to CO2 was high in enterocytes from neonatal horses, but low in cells from adult and aged horses. Enterocytes from all age groups of horses did not degrade proline into CO2. Regardless of age, equine enterocytes formed ornithine from glutamine and proline, but failed to convert ornithine into citrulline and arginine. Because arginine is an essential substrate for the synthesis of not only proteins, but also nitrogenous metabolites (e.g., nitric oxide, polyamines, and creatine), our novel findings have important implications for the nutrition, performance, and health of horses.

The amino acid arginine (Arg) is a precursor for the synthesis of multiple biological molecules including nitric oxide, polyamines, and creatine that are involved in cell proliferation, cellular remodeling, dilation of blood vessels, and phosphocreatine production for a readily available source of energy. Multipurpose capabilities of Arg have increased the interest in its effects in other species and must be evaluated in the horse. Levels of Arg are deficient in the milk of mammals such as humans, cows, sheep, and pigs, but their neonates are capable of synthesizing citrulline and Arg from glutamine and proline in the small intestine. High concentrations of Arg in milk have been observed in the horse, warranting investigation in case that the foal cannot synthesize Arg to support growth and thus rely on milk as the sole source of Arg. To date, no research has determined the endogenous production of Arg in horses to support metabolic and physiological processes; therefore, our experiment quantifies the synthesis of Arg in enterocytes of the small intestine of neonatal, adult, and aged horses. Data collected from this study serve as the necessary first step to determine the Arg requirement in the horse that has over-reaching implications to improve the growth, performance, reproductive efficiency, and to enhance longevity of the horse.

Effects of crude protein content on intake and digestion of coastal bermudagrass hay by horses.

This study was conducted to determine the effects of forage crude protein (CP) level on intake and digestion of Coastal bermudagrass hay by horses. Four cecally fistulated geldings were used in a 4 × 4 Latin square design with four treatments and four periods. Horses were fed one of four Coastal bermudagrass hays consisting of 7%, 10%, 13%, or 16% CP during each of the four 15-d periods. Intake and apparent digestibility were determined for each horse at the end of each period by total fecal collection. In addition, cecal fluid and blood samples were collected on the last day of each period for the determination of cecal ammonia, cecal pH, plasma urea nitrogen (PUN), and plasma glucose concentrations. Data were analyzed using PROC MIXED of SAS. CP concentration of Coastal bermudagrass hay influenced equine intake and digestion. Increasing CP concentration linearly increased digestible organic matter intake (DOMI) from 3.79 to 5.98 kg/d for 7% and 16% CP hay, respectively (P = 0.04). Furthermore, as the forage CP level increased, CP intake increased linearly (P < 0.01). The forage CP level had no effect on forage dry matter intake. Quadratic effects (P ≤ 0.05) were observed for forage OM, neutral detergent fiber, acid detergent fiber, and digestible energy. Overall digestibility was lowest for the 7% CP hay and highest for the 10% CP hay. Cecal pH remained above 6.62 irrespective of treatment and time, indicating that cecal pH was suitable for microbial growth. As the forage CP level increased, cecal ammonia concentration increased linearly from 0.03 mM for the 7% to 1.74 mM for the 16% CP hay (P < 0.01). Concentration of plasma glucose also linearly increased (P = 0.04) from 68.77 to 73.68 mg/dL as CP concentration increased from 7% to 16% CP. PUN exhibited a quadratic effect as concentration increased (P < 0.01) from 4.34 to 5.61 mM for the 7% and 16% CP hays, respectively. Overall, the 10% CP hay had the highest digestibility due to its higher OM digestion. As forage OMI increased, digestible OM increased until physiological capacity for digestion is exceeded. At that point, digestion will decline with the decrease in OMI, explaining the lower digestion for other forages fed.

Evaluation of an oral joint supplement on gait kinematics and biomarkers of cartilage metabolism and inflammation in mature riding horses.

Twenty stock-type horses (589 ± 126 kg BW; 13 ± 8 yr) were used in a completely randomized design for 28-d to evaluate the impact of a joint supplement on gait kinematics, inflammation, and cartilage metabolism. Horses were stratified by age, sex, body weight (BW), and initial lameness scores and were randomly assigned to one of two dietary treatments consisting of either a 100-g placebo top-dressed daily to 0.6% BW (as-fed) commercial concentrate (CON; n = 10; SafeChoice Original, Cargill, Inc.), or an oral joint supplement (SmartPak Equine LLC) containing glucosamine, chondroitin sulfate, hyaluronic acid, methylsulfonylmethane, turmeric, resveratrol, collagen, silica, and boron (TRT; n = 10). Horses were group-housed with ad libitum access to coastal bermudagrass hay (Cynodon dactylon) and allowed to graze pasture 2 h/d. Horses were exercised progressively 4 d/wk at 45 min each. On days 13 and 27, blood was harvested followed by a 19.3-km exercise stressor on concrete. Horses traveled at the walk, with no more than 15 min at the trot. Every 14 d, BW and BCS were recorded, and blood was collected for plasma prostaglandin E2 (PGE2), serum collagenase cleavage neopeptide (C2C), carboxypropeptide of type II collagen (CPII), and chondroitin sulfate 846 epitope (CS846) analysis. Kinematic gait analysis was performed every 14 d (Kinovea v.0.8.15) to determine stride length (SL) and range of motion (ROM) of the knee and hock at the walk and trot. Data were analyzed using PROC MIXED of SAS. All horses increased BW and BCS over time (P ≤ 0.01). Hock ROM increased in TRT horses (P ≤ 0.02) at the walk and tended to increase at the trot compared to CON (P = 0.09). At the walk, SL and knee ROM increased over time, independent of dietary treatment (P ≤ 0.01); no time effect was observed at the trot (P > 0.15). Regardless of treatment, C2C and CPII increased over time (P ≤ 0.05) and no effect was observed for CS846 or PGE2 (P > 0.12). In response to the exercise stressor, CPII and PGE2 decreased (P ≤ 0.05) from day 13 to 14, and CS846 and PGE2 tended to decrease (P ≤ 0.10) from day 27 to 28, independent of dietary treatment. In conclusion, hock ROM at the walk and trot was most sensitive to dietary treatment. Supplementation did not alter biomarker concentration of collagen metabolites or systemic inflammation in the 28-d period, but a future study utilizing arthrocentesis may be warranted to specifically evaluate intra-articular response to dietary treatment.

Influence of diet fortification on body composition and apparent digestion in mature horses consuming a low-quality forage.

Stock-type mares (498 ± 9 kg BW; 12 ± 7 yr) were used in a completely randomized design for 56 d to test the hypothesis that concentrate fortification improves apparent digestion and enhances lean mass over the topline. Horses were stratified by age, BW, and BCS and randomly assigned to either a custom pelleted concentrate (CON; n = 13), or an iso-caloric, iso-nitrogenous pellet that included amino acid fortification, complexed trace minerals, and fermentation metabolites (FORT; n = 10). Concentrate was offered at a total 0.75% BW/d (as-fed) twice daily, and diets were designed to meet or exceed maintenance requirements for mature horses. Horses had ad libitum access to Coastal bermudagrass hay (7.4% CP, 67% NDF, and 40% ADF). Every 14 d BW and BCS were recorded, and ultrasound images were captured every 28 d. longissimus dorsi area (LDA) and subcutaneous fat thickness (FT) were measured between the 12th and 13th ribs (12th/13th) and 17th and 18th ribs (17th/18th). Intramuscular fat at the 17th/18th ribs and rump fat-thickness were also obtained. Horses were dosed with 10 g/d of titanium dioxide (TiO2) for 14 d to estimate forage dry matter intake (DMI). To account for diurnal variation, fecal samples were collected twice daily at 12-h intervals during the last 4 days, advancing by 3 h each day to represent a 24-h period. Fecal samples were composited by horse and analyzed for TiO2 to estimate fecal output and acid detergent insoluble ash was used to calculate forage DMI. To evaluate body composition, horses were infused with a 0.12 g/kg BW deuterium oxide (D2O) on d 0 and 56. Body fat percentage (BF) was determined by quantifying D2O in plasma samples collected at pre- and 4-h postinfusion via mass spectrometry. All data were analyzed using PROC MIXED (SAS v9.4). The model contained a fixed effect of diet; horse (diet) was a random effect. Horses receiving FORT gained 17th/18th FT (P < 0.01) and increased 17th/18th LDA from d 0 to 56 (P < 0.01) while 17th/18th FT and LDA were unchanged in CON. Regardless of diet, BF estimated by D2O infusion increased in all horses from d 0 to 56 (P < 0.01). Average hay DMI was 2.1% BW, but did not differ between diets. In this study, concentrate fortification did not significantly (P ≥ 0.27) affect apparent digestion. In conclusion, concentrate fortification may promote greater muscle development along the posterior topline.

Evaluation of dietary trace mineral supplementation in young horses challenged with intra-articular lipopolysaccharide.

Sixteen weanling Quarter Horses (255 ± 22 kg) were utilized in a 56-d trial to evaluate the effects of trace mineral (TM) source on intra-articular inflammation following a single acute inflammatory insult. Horses were stratified by age, sex, and BW and then randomly assigned to dietary treatment: concentrate formulated with Zn, Mn, Cu, and Co as inorganic sources (CON; n = 8) or complexed TMs (CTM; n = 8). Added TM were formulated at iso-levels across treatments and intakes met or exceeded NRC requirements. Horses were offered 1.75% BW (as-fed) of treatment concentrate and 0.75% BW (as-fed) coastal Bermudagrass hay. Growth measurements were collected on days 0, 28, and 56, and plasma was collected biweekly for determination of Mn, Cu, Zn, and Co concentrations. On day 42, carpal joints were randomly assigned to receive injections of 0.5 ng lipopolysaccharide (LPS) or sterile lactated Ringer's solution (LRS; contralateral control). Synovial fluid was collected at preinjection hours (PIH) 0, and 6, 12, 24, 168, and 336 h post-injection and analyzed for TM concentration, prostaglandin E2 (PGE2), carboxypeptide of type II collagen (CPII), collagenase cleavage neopeptide (C2C), and aggrecan chondroitin sulfate 846 epitope (CS846). Data were analyzed using the MIXED procedure of SAS. Results showed a TM source × LPS × h effect for synovial fluid Co, Cu, and Se (P < 0.05); concentrations of TM peaked at hour 6 and decreased to preinjection values by hour 168 in both CON and CTM-LPS knees. A delayed peak was observed at hour 12 for CTM-LRS. Peak synovial fluid Cu and Se concentrations were higher in LPS knees, and Co was highest in CTM-LPS. A TM source × h interaction was observed for Zn (P < 0.05); concentrations peaked at hour 6 in CON vs. hour 12 for CTM. An LPS × h interaction was observed for Mn (P < 0.01); synovial concentration peaked at hour 6 in LPS knees compared with hour 24 in LRS. Synovial PGE2, C2C, CPII, and CS846 concentrations were greater with LPS (P ≤ 0.01), and C2C was greater (P < 0.01) in CTM compared with CON. Concentrations of CPII and PGE2 were unaffected by diet. A TM source × h × LPS interaction was observed for CS846 (P = 0.02). Concentrations of CS846 in CTM peaked at 12 h, whereas CON peaked at a lower concentration at 24 h (P < 0.05). Data indicate sufficient intake of a complexed TM source may support cartilage metabolism through increased aggrecan synthesis and type II collagen breakdown following an intra-articular LPS challenge in growing horses.

Effects of aquatic conditioning on cartilage and bone metabolism in young horses.

While beneficial in rehabilitation, aquatic exercise effects on cartilage and bone metabolism in young, healthy horses has not been well described. Therefore, 30 Quarter Horse yearlings (343 ± 28 kg; 496 ± 12 d of age) were stratified by age, body weight (BW), and sex and randomly assigned to 1 of 3 treatments for 140-d to evaluate effects of aquatic, dry, or no exercise on bone and cartilage metabolism in young horses transitioning to an advanced workload. Treatments included nonexercise control (CON; n = 10), dry treadmill (DRY; n = 10), or aquatic treadmill exercise (H2O; n = 10; water: 60% wither height, WH). Horses were housed individually (3.6 × 3.6 m) from 0600 to 1800 hours, allowed turnout (74 × 70 m) from 1800 to 0600 hours, and fed to meet or exceed requirements. During phase I (days 0 to 112), DRY and H2O walked on treadmills 30 min/d, 5 d/wk. Phase II (days 113 to 140) transitioned to an advanced workload 5 d/wk. Every 14-d, WH, hip height (HH), and BW were recorded. Left third metacarpal radiographs on days 0, 112, and 140 were analyzed for radiographic bone aluminum equivalence (RBAE). Every 28-d, serum samples were analyzed for osteocalcin and C-telopeptide crosslaps of type I collagen (CTX-1), and synovial fluid samples were analyzed for prostaglandin E2, collagenase cleavage neopeptide (C2C), collagenase of type I and type II collagen, and carboxypeptide of type II collagen using ELISAs. All data were analyzed using PROC MIXED of SAS, including random effect of horse within treatment, and repeated effect of day. Baseline treatment differences were accounted for using a covariate. There were treatment × day interactions (P < 0.01) where OC and CTX-1 remained consistent in both exercise groups while inconsistently increasing in CON. There were no treatment differences (P > 0.30) in RBAE, BW, or HH, but all increased over time (P < 0.01). There were no treatment × day interactions of synovial inflammation or markers of cartilage metabolism; however, there was an effect of day for each marker (P<0.03). Changes in biomarkers of cartilage turnover in horses exercised at the walk, whether dry or aquatic, could not be distinguished from horses with access to turnout alone. This study indicates that early forced exercise supports consistent bone metabolism necessary for uniform growth and bone development, and that there are no negative effects of buoyancy on cartilage metabolism in yearlings transitioned from aquatic exercise to a 28-d advanced workload.

Evaluation of conjugated linoleic acid supplementation on markers of joint inflammation and cartilage metabolism in young horses challenged with lipopolysaccharide.

Seventeen yearling Quarter Horses were used in a randomized complete block design for a 56-d trial to determine ability of dietary CLA to mitigate joint inflammation and alter cartilage turnover following an inflammatory insult. Horses were blocked by age, sex, and BW, and randomly assigned to dietary treatments consisting of commercial concentrate offered at 1% BW (as-fed) supplemented with either 1% soybean oil (CON; n = 6), 0.5% soybean oil and 0.5% CLA (LOW; n = 5; 55% purity; Lutalin, BASF Corp., Florham Park, NJ), or 1% CLA (HIGH; n = 6) top-dressed daily. Horses were fed individually every 12 h and offered 1% BW (as-fed) coastal bermudagrass (Cynodon dactylon) hay daily. This study was performed in 2 phases: phase I (d 0 to d 41) determined incorporation of CLA into plasma and synovial fluid; phase II (d 42 to d 56) evaluated potential of CLA to mitigate intra-articular inflammation and alter cartilage metabolism. Blood and synovial fluid were collected at 7- and 14-d intervals, respectively, to determine fatty acid concentrations. On d 42, carpal joints within each horse were randomly assigned to receive intra-articular injections of 0.5 ng lipopolysaccharide (LPS) derived from Escherichia coli 055:B5 or sterile lactated Ringer's solution. Synovial fluid samples were obtained at preinjection h 0 and 6, 12, 24, 168, and 336 h postinjection, and analyzed for prostaglandin E2 (PGE2), carboxypeptide of type II collagen (CPII), and collagenase cleavage neopeptide (C2C). Data were analyzed using PROC MIXED procedure of SAS. Horses receiving the CON diet had undetectable levels of CLA for the duration of the study. A quadratic dose response was observed in concentrations of CLA in plasma and synovial fluid (P < 0.01). A negative quadratic dose response was observed for plasma arachidonic acid (20:4) with a reduction in concentration to d 14 in HIGH horses (P = 0.04). Synovial fluid 20:4 tended to decrease in horses receiving the HIGH diet (P = 0.06). Post LPS injection, synovial PGE2 was not affected by dietary treatment (P = 0.15). Synovial C2C was lower in HIGH horses (P = 0.05), and synovial CPII tended to be greater in LOW horses than HIGH and CON horses (P = 0.10). In conclusion, dietary CLA incorporated into plasma and synovial fluid prior to LPS challenge. Dietary CLA did not influence inflammation; however, there was a reduction in cartilage degradation and an increase in cartilage regeneration.