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

Evaluation of dietary trace mineral supplementation in young horses challenged with... Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Evaluation of dietary trace mineral supplementation in young horses challenged with intra-articular lipopolysaccharide ,2 † Allison A. Millican,* Jessica L. Leatherwood,* Josie A. Coverdale,* Carolyn E. Arnold, ‡ § , # Amanda N. Bradbery,* Connie K. Larson, Emily D. Lamprecht, Sarah H. White,* Chad B. Paulk, Thomas H. Welsh, Jr,* and Tryon A. Wickersham* *Department of Animal Science, Texas A&M University, College Station, TX 77843; Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX 77843; Zinpro Corporation, Eden Prairie, § # MN 55344; Cargill Animal Nutrition, Elk River, MN 55330; and Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506 ABSTRACT:  Sixteen weanling Quarter Horses MIXED procedure of SAS. Results showed a TM (255 ± 22 kg) were utilized in a 56-d trial to evaluate source × LPS × h effect for synovial fluid Co, Cu, the effects of trace mineral (TM) source on intra-ar- and Se (P  <  0.05); concentrations of TM peaked ticular inflammation following a single acute in- at hour 6 and decreased to preinjection values by flammatory insult. Horses were stratified by age, hour 168 in both CON and CTM–LPS knees. A de- sex, and BW and then randomly assigned to dietary layed peak was observed at hour 12 for CTM–LRS. treatment: concentrate formulated with Zn, Mn, Peak synovial fluid Cu and Se concentrations were Cu, and Co as inorganic sources (CON; n = 8) or higher in LPS knees, and Co was highest in CTM– complexed TMs (CTM; n = 8). Added TM were for- LPS. A TM source × h interaction was observed for mulated at iso-levels across treatments and intakes Zn (P < 0.05); concentrations peaked at hour 6 in met or exceeded NRC requirements. Horses were CON vs. hour 12 for CTM. An LPS × h interaction offered 1.75% BW (as-fed) of treatment concentrate was observed for Mn (P < 0.01); synovial concen- and 0.75% BW (as-fed) coastal Bermudagrass hay. tration peaked at hour 6 in LPS knees compared Growth measurements were collected on days 0, 28, with hour 24 in LRS. Synovial PGE , C2C, CPII, and 56, and plasma was collected biweekly for de- and CS846 concentrations were greater with LPS (P termination of Mn, Cu, Zn, and Co concentrations. ≤ 0.01), and C2C was greater (P  <  0.01) in CTM On day 42, carpal joints were randomly assigned compared with CON. Concentrations of CPII and to receive injections of 0.5  ng lipopolysaccharide PGE were unaffected by diet. A TM source × h × (LPS) or sterile lactated Ringer’s solution (LRS; LPS interaction was observed for CS846 (P = 0.02). contralateral control). Synovial fluid was collected Concentrations of CS846 in CTM peaked at 12 h, at preinjection hours (PIH) 0, and 6, 12, 24, 168, whereas CON peaked at a lower concentration at and 336 h post-injection and analyzed for TM con- 24  h (P  <  0.05). Data indicate sufficient intake of centration, prostaglandin E (PGE ), carboxypep- a complexed TM source may support cartilage me- 2 2 tide of type II collagen (CPII), collagenase cleavage tabolism through increased aggrecan synthesis and neopeptide (C2C), and aggrecan chondroitin sulfate type II collagen breakdown following an intra-artic- 846 epitope (CS846). Data were analyzed using the ular LPS challenge in growing horses. Key words: equine, inflammation, lipopolysaccharide, trace minerals 1 2 The authors affirmatively acknowledge that they were Corresponding author: leatherwood@tamu.edu free from influence by Zinpro Corporation and its employ- Received October 3, 2019. ees that would result in any conflict of interest. The authors Accepted January 14, 2020. acknowledge the labor and efforts provided on this project by fellow graduate students: K. Fikes, M. S. Goehring, E. C. Dicksen, C. M. Latham, and C. J. Hartz. 1 Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. © The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non- Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commer- cial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Transl. Anim. Sci. 2020.4:1-16 doi: 10.1093/tas/txaa006 INTRODUCTION MATERIALS AND METHODS Homeostatic maintenance of articulating All care, handling, and procedures for experi- joints requires Cu, Mn, and Zn for turnover of col- ment were approved by the Texas A&M University lagen fibrils and to contribute to molecules within Institutional Animal Care and Use Committee. the extracellular matrix (Hostetler et  al., 2003; Richards et  al., 2010). The role of dietary trace Horses and Treatments minerals (TM) in equine articulating joints remains undetermined; however, information gained from Sixteen weanling Quarter Horses (mean ± other species indicates that metal amino acid-com- SEM; initial BW of 255 ± 22 kg BW; n = 9 colts; plexed TM provide a more biologically available n  =  7 fillies) were used in a complete randomized source of TM (Osorio et  al., 2012). In hens chal- design. Prior to the initiation of dietary treatments, lenged with systemic lipopolysaccharide (LPS) and mares and foals were maintained on the same com- supplemented with Zn amino acid complex, serum mercial concentrate that contained inorganic min- interleukin-1β (IL-1β) concentration increased to eral sources (Producer’s Cooperative Association, 3 h post-induction, but at 12 h, concentrations were Bryan, TX). The concentrate was provided as a lower than hens receiving Zn sulfate (Cheng and creep feed to foals beginning at 90 d of age. Foals Guo, 2004). were weaned at 150 ± 11 d of age and maintained Increased bioavailability and utilization of on the same concentrate until the initiation of the complexed TM may allow for greater incorporation study (233 ± 20 d of age). of TM into articular cartilage and a more rapid Radiographs (lateral, flexed lateral, and crani- achievement in homeostasis following endotoxin al-caudal views) of both radial carpal joints were injection. Utilizing an intra-articular LPS challenge performed at the Texas A&M University Large to induce localized inflammation and cartilage turn- Animal Hospital (College Station, TX) prior to over in young horses provides the ability to evaluate initiation of the study. All horses considered to be the effect of TM source on cartilage metabolism, radiologically normal by a licensed veterinarian joint inflammation, and synovial fluid TM concen- were stratified by age, sex, BW, and BCS, and ran- trations post-induction (Leatherwood et  al., 2016) domly assigned to dietary treatment. Treatment through biomarkers relative to inflammation and diets consisted of isocaloric, isonitrogenous pel- cartilage turnover. leted concentrate formulated with either inorganic Synovial prostaglandin E (PGE ) concen- (CON; 100% inorganic CuSO , ZnSO , and MnSO 2 2 4 4 4 tration increases in response to intra-articular and CoCO ; n = 8) or TM complexes (CTM; zinc LPS. Resulting inflammation influences collagen methionine, manganese methionine, copper lysine, metabolism and aggrecan synthesis by increas- and cobalt glucoheptonate; n = 8). Added levels of ing catabolic collagenase cleavage neopeptide Zn, Mn, Cu, and Co were formulated at iso-levels (C2C), anabolic carboxypropetide of type II col- in both pelleted treatment diets, and daily mineral lagen (CPII), and chondroitin sulfate 846 epitope intakes met or exceeded 2007 NRC minimum re- (CS846; de Grauw et al., 2009; Lucia et al., 2013). commended requirements. All personnel involved Therefore, objectives of this experiment were to in performing the study were blinded to dietary compare effects of dietary TM source (organic treatment. Composited samples of concentrate and vs. inorganic) on growth, joint inflammation, car - hay were analyzed by Equi-Analytical Laboratories tilage metabolism, and synovial fluid TM con- (Ithaca, NY) commercial analysis (Table  1) for centrations in response to an intra-articular LPS dry matter (method 930.15; AOAC, 2019), crude challenge in young horses. petrol (method 990.03; AOAC, 2019), crude fat Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses Table 1. Nutrient composition of concentrates and on days 0, 28, and 56 by the same 3 independent hay (DM basis) fed to weanling horses observers. Rump fat was measured at 5  cm lateral from the midline, halfway between the first coccy- Concentrate geal vertebrae and the ischium (Westervelt et  al., 1 2 3 CON CTM Forage 1976). An altitude stick was used to measure WH Dry matter, % 89.50 91.3 89.16 and HH. Body length and HG were taken using a Nutrient, % DM soft measuring tape. Ultrasonic images were also CP 19.30 19.6 12.51 captured of the longissimus dorsi muscle (LM) were ADF 25.90 23.7 35.51 captured by a certified technician (Designer Genes NDF 40.00 36.1 65.99 Technologies, Inc., Harrison, AR) to determine Fat 7.70 7.9 2.26 Ca 0.94 1.26 0.32 muscle area, back fat thickness (BFT), and intra- P 0.92 1.16 0.29 muscular fat (IMF) deposition. The transducer was Trace minerals , ppm placed to obtain a cross-sectional image taken be- Zn 197.90 184.7 17.9 tween the 13th and 14th as well as the 17th and 18th Cu 51.90 57.9 5.8 ribs. Subcutaneous fat thickness was measured at Mn 216.00 226.9 192.2 three-fourths the distance from the medial end of Co 7.10 9.5 <1.0 the LM; 4 independent images were collected lat- Se 0.73 0.89 erally across the 17th and 18th ribs to estimate IMF Control: pelleted concentrate formulated with 100% inorganic min- within the LM. Four independent images were ne- eral sources (CuSO , ZnSO , and MnSO and CoCO ), supplied for 56 4 4 4 3 cessary to follow Annual Proficiency Testing and d at 1.75% BW (as-fed), n = 8. Certification standard format for data submission. Trace mineral complexes: 7  g of 4-Plex C (Zinpro Corporation, Proper contact between transducer and horse was Eden Prairie, MN) replaced a portion of inorganic added trace min- eral, supplied for 56 d at 1.75% BW (as-fed), n = 8. insured by fitting the transducer with a PIA con- Coastal bermudagrass, Cynodon dactylon, supplied to both treat- tour pad (Animal Ultrasound Services, Ithaca, NY) ment groups at 0.75% BW. designed to conform to the curvature of the horse’s Total mineral content. back. In addition, corn oil was applied to promote Reported on a 100% dry basis. acoustical contact between animal and transducer (Perkins et  al., 1992). An independent laboratory (method 2003.05; AOAC, 2019), acid detergent interpreted all images; personnel were blinded to fiber (ANKOM Technology Method 5), neutral de- treatment (Designer Genes Technologies, Inc.). tergent fiber (ANKOM Technology Method 6), Ca, and P (inductively coupled plasma analysis [ICP]), Incorporation into Plasma and Synovial Fluid and TM concentrations (Fe, Zn, Cu, Mn, Mo, Co, Plasma samples for TM analysis were collected Se) were analyzed by Michigan State University every 14 d into a 6-mL trace element K EDTA, DCPAH (Lansing, MI) with an ICP/mass spec- 10.8  mg, additive tube (BD Vacutainer, Franklin trometer (ICP/MS). Lakes, NJ) prior to the morning feeding. Samples Weanlings received their respective pelleted were immediately placed on ice until centrifugation concentrate at 1.75% BW/d (as-fed) and 0.75% at 2,000 × g for 10 min at 4 °C within 1 h of collec- BW/d (as-fed) of coastal bermudagrass (Cynodon tion. After centrifugation, plasma was aliquoted dactylon) hay divided evenly between 2 feedings at into 1.5-mL microcentrifuge tubes and stored at 0600 and 1800. Horses were fed individually and −80 °C until analysis. A certified veterinarian from maintained in 3 × 3 m stalls with ad libitum access the Texas A&M University Large Animal Clinic to water. Intakes and orts were obtained and meas- (Dr. C. E. Arnold) performed carpal arthrocente- ured daily. Every 7 d, BW was obtained utilizing sis on both radial carpal joints on day 0.  Horses a calibrated digital platform scale (Bastrop Scale were sedated using xylazine HCl, administered Inc., Bastrop, TX) and diets adjusted accordingly. intravenously at recommended dosages. The All horses were allowed 8 h of free exercise in a dry carpal joint was aseptically aspirated using a loca- lot (58.5 × 79.2 m) daily. tion medial to the extensor carpi radialis tendon in the palpable depression between the radial carpal Growth and Performance Characteristics bone and the third carpal bone, to a depth of ap- Body condition score, rump fat (RF), wither proximately 12.7 mm to avoid unnecessary contact height (WH), hip height (HH), body length (BL), with articular cartilage (McIlwraith and Trotter, and heart girth circumference (HG) were taken 1996). Pooled synovial fluid between carpal joints Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. (1 to 4  mL) was transferred into sterile nonaddi- Sample Analysis tive tubes (BD Vacutainer) and was immediately Plasma and synovial fluid samples were sent placed on ice and stored at −80 °C until laboratory to the Michigan State Diagnostics Laboratory analysis. (Lansing, MI) for TM analysis to establish TM composition. In brief, samples were diluted 20-fold Intra-articular LPS Challenge with a solution containing 0.5% EDTA and Triton On day 42 of the study, all horses were sub- X-100, 1% ammonium hydroxide, 2% propanol, jected to an intra-articular LPS challenge. One and 20 ppb of scandium, rhodium, indium, and radiocarpal joint was randomly selected within bismuth as internal standards. The ICP/MS was each horse for injection with LPS, whereas the tuned to yield a minimum of 7,500 cps sensitivity other radiocarpal joint served as a contralateral for 1 ppb yttrium (mass 89), less than 1.0% oxide control (injection of sterile lactated Ringer’s so- level as determined by the 156/140 mass ratio lution; LRS). The use of an LRS joint was based and less than 2.0% double charged ions as deter- on previous data in our laboratory that suggested mined by the 70/140 mass ratio (Wahlen et  al., repeated arthrocentesis influenced local inflam- 2005). Elemental concentrations were calibrated matory status in horses regardless of treatment using a 4-point linear curve of the analyte-inter- (LRS or LPS) as evidenced by an alteration in nal standard response ratio. Standards were from circulating leukocytes, monocytes, or platelets Inorganic Ventures (Christiansburg, VA). In-house (Hunt et al., 2018). serum pools were used as controls. At PIH 0, the carpal joint was aseptically pre- Synovial fluid concentrations CPII, C2C, and pared for arthrocentesis and horses were sedated CS846 were measured using commercially avail- as previously described. Purified LPS derived from able ELISA kits (IBEX Pharmaceuticals Inc., Escherichia coli O55:B5 (Sigma Aldrich, St. Louis, Montreal, QC, Canada) previously validated in MO) was reconstituted and diluted in sterile lac- horses (de Grauw et al., 2009; Lucia et al., 2013). tated Ringer’s solution; individual doses were Synovial fluid samples were analyzed in duplicate, 0.8  mL with a final concentration of 0.5  ng/mL. and standards were prepared according to man- Dosage of LPS was based on previous work in our ufacturer’s recommendations with samples pre- laboratory (Lucia et al., 2013; Leatherwood et al., pared at a 1:4 dilution for both CPII and C2C. 2016; Kahn et al., 2017). The LPS solution was in- Sample dilutions for CS846 ranged from 1:50 serted aseptically into the randomly selected carpal to 1:1,000 depending on time post-injection, to joint, and LRS joints were injected with 0.8  mL remain within detectable limits of the ELISA. of sterile lactated Ringer’s solution after the with- Dilutions were made with calibrator diluents drawal of the PIH 0 sample. Synovial fluid samples provided by the kit prior to beginning the assay. (1 to 4 mL) were obtained at PIH 0 and 6, 12, 24, Mean detectable concentrations for CPII, C2C, 168, and 336 h post-injection). All personnel were and CS846 were 50, 10, and 20  ng/mL, respect- blinded to injection type. ively. Shifts in cartilage metabolism were evaluated All synovial samples were collected and trans- by the ratio of CPII to C2C with individual ratios ferred to sterile nonadditive tubes (BD Vacutainer calculated for each horse. Blood Serum Collection Tubes; Becton-Dickinson Synovial fluid samples were analyzed in du- and Company, Franklin Lakes, NJ) and imme- plicate for concentrations of PGE utilizing an diately placed on ice until aliquoted into 1.5-mL enzyme-linked immunoassay (R&D Systems, microcentrifuge tubes. Aliquots were stored at Minneapolis, MN), previously validated in horses −80  °C until later analysis of C2C, CPII, CS846, (Bertone et al., 2001; de Grauw et al., 2006; Lucia PGE , and TM concentrations. All horses were et  al., 2013). Samples intended for PGE analysis 2 2 monitored for signs of anaphylaxis over the initial were diluted from 1:1 to 1:10 depending on time 24  h post-injection. Rectal temperature (RT; °C), post-injection to remain within detectable limits of heart rate (HR; beats/min), and respiratory rate the ELISA; dilutions were prepared using the cali- (RR; breaths/min) were recorded prior to arthro- brator diluent provided by the kit with a mean de- centesis at PIH 0 and at 6, 12, and 24 h post-injec- tectable dose of PGE of 39 pg/mL. tion. Carpal circumference (cm) was measured at Final concentrations of all markers were read the level of the accessory carpal bone with a soft using a microplate reader (Synergy H1, Biotek, tape measure that was performed by a single indi- Winooski, VT) with optical density set at 450 nm. vidual to maintain consistency. Intracoefficients of variation for CPII, C2C, and Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses PGE were less than or equal to 15% and less than evaluate normal distributions for traits based on 20% for CS846. continuous variables. One biomarker (CPII) exhib- ited non-normal data; therefore, the data were log transformed for normalization. Outliers were iden- Statistical Analysis tified using box plots of the residuals and removed Intake data were analyzed by using the MIXED if greater than 3 SD from the mean. Data are re- procedure of SAS v9.4 (SAS Inst. Inc., Cary, NC). ported as mean ± SEM. The model contained fixed effects of TM source and time (d). Initial, final, and delta values for growth RESULTS parameters were analyzed using the MIXED pro- cedure of SAS v9.4 (SAS Inst. Inc., Cary, NC). The Target intakes of 1.75% BW (as-fed) from con- model contained a fixed effect of TM source and centrate and 0.75% BW (as-fed) from hay were used a random variable of horse within treatment. achieved as horses consumed their respective diets Plasma mineral concentration utilized the same with no significant refusals across treatment groups model with an additional fixed effect of time (d) over the 56-d trial. Weanling horses consumed en- and a TM source × d interaction. ergy and associated nutrients to meet or exceed re- In response to the LPS challenge, statistical commended requirements (NRC, 2007). Total dry analysis of synovial fluid biomarker and TM con- matter intake throughout the 56-d study did not centration were analyzed using PROC MIXED of differ between TM source (P = 0.89), with daily in- SAS v9.4 (SAS Inst. Inc., Cary, NC). The model takes of 5.74 and 5.71 ± 0.15 kg for CON and CTM, contained fixed effects of TM source, time (h), respectively. Individually, concentrate and forage injection type (LPS), and their respective inter- intakes were not different between TM sources actions. This model included a random effect of (P = 0.54 and P = 0.47, respectively). Similarly, TM horse within treatment and a repeated variable source did not affect (P = 0.78) final BW (285.8 and (time). The covariate structure was utilized to 288.8 ± 5.57 kg for CON and CTM, respectively), specify a random effect for differences between with all horses gaining 32  ± 2  kg during the 56 d animals within treatment, creating a correlation (P = 0.90), validating the diets were isocaloric. structure within animals that decreases with the No effect of TM source was observed in syn- increasing amount of time between measurements ovial biomarker concentrations during the pre-LPS (Littell et  al., 1998). The model for synovial fluid dietary adaptation period; however, a TM source TM analysis also included hour 0 as a covariate. × d interaction was observed for CS846 (P = 0.05; Post hoc comparison of TM source and individual Table  2). Synovial fluid CS846 concentration in- time points was conducted using a paired t-test. creased from days 0 to 42 in CTM horses, while Significant differences were declared as P ≤ 0.05, remaining similar from day 0 to day 42 in CON and P ≤ 0.10 was considered a trend toward sig- horses. Conversely, the ratio of CPII to C2C in- nificance. Plots of residual variation were used to creased in all horses from days 0 to 42 (P  =  0.04), Table 2.  Cartilage biomarkers and inflammatory markers within the synovial fluid of weanling Quarter Horses before (day 0) and (day 42) 42 d of receiving a pelleted concentrate at 1.75% BW (as-fed) containing either inorganic (CON; n = 8) or organic (CTM; n = 8) dietary trace mineral supplementation Dietary treatments CON CTM P-value Synovial fluid variable Day 0 Day 42 Day 0 Day 42 SEM TM source d TM source × d CPII, ng/mL 764.65 967.17 713.96 988.23 231.33 0.96 0.13 0.82 C2C, ng/mL 270.93 260.86 276.66 297.89 29.23 0.48 0.80 0.49 CPII:C2C 2.76 3.82 2.54 3.90 0.91 0.95 0.04 0.80 a a a b CS846, ng/mL 23,820 24,803 19,701 29,717 3,411 0.92 0.02 0.05 PGE , pg/mL 596.85 336.37 750.68 423.02 87.90 0.13 <0.01 0.64 Control (CON): pelleted concentrate formulated with 100% inorganic mineral sources (CuSO , ZnSO , and MnSO and CoCO ), supplied at 4 4 4 3 1.75% BW (as-fed), n = 8. Trace mineral complexes (CTM): 7 g of 4-Plex C (Zinpro Corporation, Eden Prairie, MN) replaced a portion of inor- ganic added trace mineral, supplied at 1.75% BW (as-fed), n = 8. 2 ; ; CPII = carboxypropetide of type II collagen; C2C = collagenase cleavage neopeptide CS846 = chondroitin sulfate 846 epitope PGE  = pros- taglandin E . ab Within row, means with different letters differ (P < 0.05). Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. whereas PGE decreased over time (P  <  0.01), re- Table 3.  Growth parameters and composition of gardless of TM source (Table 2). weanling Quarter Horses before (day 0)  and after 56 d of receiving a pelleted concentrate at 1.75% BW (as-fed) containing either inorganic (CON; Growth Metrics n = 8) or organic (CTM; n = 8) dietary trace min- eral supplementation Growth measurements including WH, HH, HG, and BL were not affected (P ≥ 0.56) by TM Dietary source (Table  3). Similarly, no differences between treatments TM sources were observed for final IMF, BFT, or 1 2 Variable CON CTM SEM P-value rump fat (P > 0.10). However, final BCS tended to BW, kg be lower (P = 0.06) for horses fed CTM when com- Day 0 256.4 253.7 8.0 0.82 Day 56 288.8 285.8 7.6 0.78 pared with CON. Final LM area at both measured Wither height, cm locations was not affected by TM source (P ≥ 0.60). Day 0 128.19 127.56 0.97 0.65 Overall change in area at the 17th and 18th rib was Day 56 131.68 131.60 0.67 0.93 unaffected by TM source, but a positive change Hip height, cm in area over the 56-d trial was observed in CON Day 0 134.54 134.38 1.12 0.92 2 2 (+1.72 cm ) and CTM treatments (+1.97 cm ), indi- Day 56 137.80 138.19 0.99 0.78 cating growth. Heart girth, cm Day 0 143.25 141.67 1.97 0.56 Plasma Mineral Concentration Day 56 149.52 148.13 1.82 0.60 Body length, cm No significant interaction or main effects of Day 0 135.21 134.94 1.39 0.89 TM source and d were observed for Mn (P ≥ 0.18) Day 56 144.74 145.31 1.35 0.77 with average concentrations of 1.73 and 1.62  ± Body condition score Day 0 5.65 5.52 0.16 0.58 0.09  ng/mL for CTM and CON, respectively. No Day 56 6.55 6.08 0.16 0.06 interaction or effect of TM was observed for Cu Rump fat (P ≥ 0.41); however, an effect of day (P < 0.01) was Day 0 0.14 0.15 0.006 0.79 present. Plasma Cu concentrations increased from Day 56 0.16 0.16 0.005 0.86 days 0 to 42 (P < 0.01; 1.07 to 1.22 ± 0.05 μg/mL for Intramuscular fat, % days 0 and 42, respectively) and remained elevated Day 0 3.62 3.65 0.17 0.91 through day 56. A TM source × d interaction was Day 56 3.64 3.48 0.17 0.52 present for Zn (P  =  0.02; Fig.  1). Concentrations Back fat thickness, 13th and 14th rib decreased in CON from days 0 to 56 while increas- Day 0 0.17 0.15 0.02 0.42 ing in CTM from day 14 to day 28 before declining Day 56 0.16 0.17 0.01 0.57 to levels similar to CON by day 42. A  significant Back fat thickness, 17th and 18th rib TM source × d interaction was observed for Co Day 0 0.14 0.14 0.01 0.74 Day 56 0.14 0.14 0.01 0.81 concentrations (P  <  0.01; Fig.  2). Cobalt concen- LM area, cm , 13th and 14th rib trations increased from days 0 to 14 for all horses Day 0 10.57 10.27 0.45 0.65 regardless of TM source; however, horses receiving Day 56 11.75 12.00 0.59 0.77 CTM had greater Co plasma concentrations com- LM area, cm , 17th and 18th rib pared with the control CON, beginning at day 14 Day 0 12.01 11.63 0.52 0.61 through day 56 of the study. Day 56 13.73 13.60 0.28 0.60 Control: pelleted concentrate formulated with 100% inorganic min- LPS Challenge eral sources (CuSO , ZnSO , and MnSO and CoCO ), supplied for 56 4 4 4 3 d at 1.75% BW (as-fed), n = 8. Clinical assessment  During the LPS chal- Trace mineral complexes: 7  g of 4-Plex C (Zinpro Corporation, lenge, no TM source × h interactions were present Eden Prairie, MN) replaced a portion of inorganic added trace min- for clinical parameters, including HR, RR, and eral, supplied for 56 d at 1.75% BW (as-fed), n = 8. Rump fat thickness measured by ultrasound. RT (P ≥ 0.33; data not shown). Average beats per minute were 48.4 and 50.9  ± 21.6 (P ≥ 0.22), for CON and CTM horses, respectively. Respiration PIH 0 levels at hour 12 (16  ± 2 breaths/min) and rate was affected by hour (P  <  0.01), with an in- hour 24 (15  ± 2 breaths/min). An effect of h was crease from PIH 0 (20 ± 2 breaths/min) to hour 6 also observed for RT (P  =  0.01). Rectal tempera- (24  ± 2 breaths/min), before decreasing to below ture increased from PIH 0 (38.0 ± 0.1 °C) to 38.4 ± Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses Figure 1. Mean plasma Zn concentrations over 56-d trial in weanling horses receiving a pelleted concentrate at 1.75% BW (as-fed) containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc methionine, manganese methionine, 4 4 4 3 copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Trace mineral source × d interaction (P < 0.05). *CTM greater than CON at day 28 (P < 0.05). Figure 2. Mean plasma Co concentrations over 56-d trial in weanling horses receiving a pelleted concentrate fed at 1.75% BW (as-fed) con- taining either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes Zn methionine, Mn methionine, 4 4 4 3 Cu lysine, and Co glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Trace mineral source × d interaction (P < 0.01). *CTM higher than CON (P < 0.01). 0.1  °C at hour 6 before returning to baseline by addition, at hour 12, Co was greater in CTM–CON hour 24 (38.0 ± 0.1 °C). All values remained within than CON–LPS and CON–LRS (P < 0.01). normal physiological limits throughout the 24-h A TM source × LPS × h effect was observed period. An LPS × h interaction was observed for for Cu (P < 0.01; Fig. 4). Concentrations of Cu in- carpal circumference (P < 0.01), as the LPS injected creased in both LPS and CON–LRS to peak con- knees increased to hour 12 and remained elevated centrations by hour 6 then decreased to hour 168, to hour 24, whereas the LRS knee increased in cir- whereas concentration of Cu was lowest at hour 6 cumference to hour 12 and returned to baseline by in CTM–LRS (P  =  0.01). Concentrations of Cu hour 24. Carpal circumference increased over time in CTM–LRS increased from hours 6 to 12 and (P < 0.01; data not shown), but was not affected by then declined to hour 168. Peak concentration for TM source. CTM–LRS was observed at hour 12, and the Cu Synovial fluid mineral concentration  A TM concentration was similar to other knees at that source × LPS × h interaction was observed for Co time point. No differences were observed at hour (P  <  0.01; Fig.  3). Cobalt concentration at hour 168 or 336. Trace mineral source × LPS × h effect 6 was the greatest in CTM–LPS (P  <  0.01). In was also observed for Se (P  =  0.02; Fig.  5), with Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. Figure 3. Mean synovial fluid cobalt concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc methionine, 4 4 4 3 manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pel- leted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Cobalt concentrations: trace mineral source × LPS abc × h interaction (P < 0.01). Denotes differences within each time point between trace mineral source and LPS (P < 0.05). Figure 4. Mean synovial fluid copper concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc methionine, 4 4 4 3 manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pel- leted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Copper concentrations: trace mineral source × LPS abc × h interaction (P = 0.01). Differences within each time point between trace mineral source and LPS (P < 0.05). concentrations following the same basic pattern CON and hour 12 for CTM before decreasing to as Cu. Concentrations of Se were lowest in CTM– hour 168. At hours 12, 24, and 168, CON had a LRS at hour 6 vs. other knees (P < 0.01) and then lower concentration of Zn than CTM (P  <  0.01). increased to similar concentrations at hour 12 to An LPS × h interaction (P  <  0.01) was observed those in other knees. Concentrations of Se then for Zn; concentrations were higher in LPS knees at declined in all knees to hour 168, and maintained hours 6 and 24 (P ≤ 0.01) and tended to be higher at similar concentrations to hour 336. No TM source hour 12 than CON (P = 0.06). × LPS × h interaction was present for Zn (P = 0.21; No TM source × h × LPS interaction (P = 0.74; Fig. 6), although concentrations followed the same Fig. 7) was observed for Mn; however, an h × LPS general pattern as Cu and Se. A  TM source × h interaction was present (P < 0.01). Manganese con- interaction was observed for Zn (P  =  0.02). Zinc centration increased in both LPS knees to hour 6 increased in concentration, peaking at hour 6 in and then decreased steadily to baseline levels by Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses Figure 5. Mean synovial fluid selenium concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc methionine, 4 4 4 3 manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Selenium concentrations: trace mineral source × abc LPS × h interaction (P = 0.02). Differences within each time point between trace mineral source and LPS (P < 0.05). Figure 6. Mean synovial fluid zinc concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc methionine, 4 4 4 3 manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pel- leted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Zinc concentrations: trace mineral source × LPS × h (P = 0.21), trace mineral source × h (P < 0.05), h × LPS (P < 0.01). *Differences within each time point between trace mineral source (P < 0.05). Differences within each time point between injection type (LPS). hour 168. LRS knees displayed an increase from Cartilage markers  An LPS × h inter- hour 12 to 24 resulting in higher Mn concentra- action was present for synovial C2C (P  <  0.01; tion in LRS compared with LPS knees at hour 24 Fig.  9). Concentrations of C2C in LPS knees (P < 0.01). Concentration of Mn in LRS knees then were greater than concentrations in LRS knees decreased to baseline levels by hour 168. at hours 6, 12, 24, and 168 (P ≤ 0.04). A  ten- Synovial inflammation  An LPS × h interaction dency for an interaction of TM source × LPS (P ≤ 0.01; Fig. 8) was observed for synovial PGE . (P  =  0.09) was observed with concentrations Concentration of PGE was greater in the LPS in- of C2C being greater in CTM–LPS compared jected knee when compared with LRS at hours 6, with CON–LPS (P  <  0.01). An LPS × h inter- 12, and 24 (P  <  0.01). Synovial PGE was not af- action (P ≤ 0.01; Fig.  10) was observed for fected by TM source (P = 0.13). anabolic CPII with LPS injection resulting in Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. Figure 7. Mean synovial fluid manganese concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments consisted of pel- leted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc me- 4 4 4 3 thionine, manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Manganese concentrations: trace mineral source × h × LPS interaction (P = 0.74), LPS × h interaction (P < 0.01), trace mineral source (P = 0.35). *Differences between LRS knees and LPS knees (P < 0.01). Figure 8. Mean synovial prostaglandin E (PGE ) concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from 2 2 Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments con- sisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes 4 4 4 3 zinc methionine, manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Trace mineral source × LPS × h (P = 0.48), LPS × h interaction (P < 0.01), trace mineral source (P = 0.13). greater CPII concentrations at 6, 12, and 168  h in LPS knees than LRS knees with no difference (P ≤ 0.01) compared with LRS. The CPII to C2C between dietary mineral sources. At hour 12, knees ratio was not significantly affected by TM source injected with LPS in CTM horses had the greatest (P  =  0.57), LPS (P  =  0.11), or hour (P  =  0.19; concentration of CS846 (P < 0.01) followed by LPS data not shown). knees in CON horses; LRS knees remained near A significant interaction of TM source × LPS baseline. By hour 24, LPS knees in CON horses × h (P  =  0.02; Fig.  11) was observed for synovial had higher CS846 concentrations than CTM horses CS846 concentrations. Prior to injection of LPS and LRS knees had increased above baseline but (PIH 0)  there were no differences in CS846 levels; remained lower than LPS. By hour 168, no differ- however, 6 h after LPS injection, CS846 was higher ences were detected. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses Figure 9. Mean synovial collagenase cleavage neopeptide (C2C) concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treat- ments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral 4 4 4 3 complexes zinc methionine, manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Trace mineral source × LPS × h (P = 0.35), LPS × h (P < 0.01), trace mineral source × LPS (P = 0.09). Figure 10. Mean synovial carboxypeptide of type II collagen (CPII) concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treat- ments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral 4 4 4 3 complexes zinc methionine, manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Trace mineral source × LPS × h (P = 0.99), LPS × h (P < 0.01), trace mineral source (P = 0.82). DISCUSSION complex has been shown to increase long bone length and width in embryos and chicks (Favero The present study examined the effect of com- et al., 2013). Limited data exist observing the effect plexed Zn, Cu, Mn, and Co source on growth and of complexed TMs on equine growth; however, intra-articular inflammation in growing horses. differences between TM proteinates and inorganic Our study did not detect a positive effect of CTM sources were evaluated in yearling horses fed for on growth of yearling horses, which is in contrast 112 d (Ott and Johnson, 2001). No effect of source with the prior reports that complexed TM in- on BW, WH, HG, or BL gains was reported; how- creased growth in beef cattle and calves (Osorio ever, HH gain was greater for horses receiving pro- et  al., 2012; Genther-Schroeder et  al., 2016a, b). tienates than the inorganic supplemented horses. Osorio et  al. (2012) reported that wither height In the current 56-d study, all horses, regardless was increased in calves fed complexed TMs (Zn, of diet, increased in BW, HH, WH, BL, and HG Mn, Cu, and Co) vs. inorganic sources at weaning. circumference. In addition, the addition of dietary Zn amino acid Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. Figure 11. Mean synovial chondroitin sulfate epitope 846 (CS846) concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treat- ments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral 4 4 4 3 complexes zinc methionine, manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Significant interactions: abc TM source × LPS × h interaction (P < 0.02). Differences in concentration among TM sources at specific time points post-intra-articular injection (P < 0.05). In previous studies exposing young horses to of each mineral increased to 6 h before returning to intra-articular LPS, clinical responses for HR, RR, baseline or below baseline levels by 168 h post-injec- and RT showed no signs of systemic illness (de tion in all knees except CTM–LRS, which showed a Grauw et al., 2009; Lucia et al., 2013). In the pre- delayed increase at 12 h. sent study, clinical measures also remained within In human osteoarthritic patients, similar in- normal physiological ranges for horses of this age creases in synovial fluid Cu and positive correl- and demonstrated the inflammatory response re- ations between synovial fluid Zn and Cu have been mained localized. Joint circumference increased reported in response to inflammation (Yazar et al., regardless of TM source or intra-articular treat- 2005). The increases in concentrations of Zn, Cu, ment (LPS vs. LRS) above baseline values at 6  h, and Se in response to LPS could be due to their increased at 12  h, and began decreasing at 24  h; role in combating free radical formation. The pres- however, values did not return to baseline by 336 h. ence of reactive oxygen species is damaging to The lack of differences between joints receiving the extracellular matric (ECM) both structurally LPS vs. LRS further validates the inclusion of and functionally (Henrotin et  al., 2003). Together, an LRS sham-injected knee in the LPS model to Se-containing glutathione peroxidase and super- account for effects of repeated arthrocentesis. Data oxide dismutase (SOD) are the major antioxidant collected in the present study agree with previous defense systems against oxygen free radicals. Three literature, indicating 0.5 ng LPS causes acute syno- isoforms of SOD exist in mammals: cytoplasmic vitis resulting in minor carpal circumference in- Cu/Zn SOD, mitochondrial Mn SOD, and extracel- creases with minimal physiological changes of HR, lular Cu/Zn SOD (Fukai and Ushio-Fukai, 2011). RR, and RT (Lucia et al., 2013; Kahn et al., 2017). Decreased SOD activity is exhibited in humans and Synovial fluid TM concentrations in response mice with osteoarthritis (Regan et  al., 2005). The to an inflammatory insult have not previously been increase in Cu could potentially be due to an in- reported in horses. Due to roles for various TMs as crease in the Cu-containing acute phase protein, enzyme activators and cofactors, changes in concen- ceruloplasmin, which appears to exert antioxidant trations have potential to affect other biochemical effects in human knee-joint synovial fluid (Blake indicators. In the present study, all TMs measured et al., 1981). varied over time, likely a result of inflammation as- In contrast to Zn, Cu, and Se, synovial fluid sociated with both LPS and repeated arthrocentesis. concentration of Mn was minimally responsive Trace minerals, Zn, Cu, Mn, and Se, were directly to LPS. Specifically, manganese concentrations of influenced by LPS injection, increasing in the syn- 6.41 and 4.81  ng/mL were exhibited in the CTM ovial fluid post-injection. Concentrations of Cu, and CON–LRS knees, respectively, at 24  h. These Zn, and Se responded similarly as concentrations values are approximately 7- to 10-fold higher than Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses values at hour 0, whereas Mn values in LPS knees interaction networks of TM under inflammatory were only 2 to 2.5 times greater (CTM and CON, conditions within the joint. Furthermore, research respectively) than hour 0. The decrease in Mn in the identifying the source of increased TM in equine LPS knees could be attributed to increased chon- synovial fluid would provide improved under - droitin sulfate synthesis, supported by the increase standing of how nutritional mineral status and car- in CS846 in response to the LPS. tilage health could result in mineral extraction from The 3-way interactions observed between diet, articular cartilage. h, and LPS for Cu and Se indicate dietary source af- Articular cartilage integrity is heavily dependent fected degree of response between knees. The peak on the balance of metabolic activities (Mueller and concentrations were highest in the CTM–LPS knee Tuan, 2011). The presence of inflammation causes for Cu and Se, 0.99  ± 0.07  μg/mL and 228.62  ± altered cartilage metabolism by decreasing anabolic 14.2 ng/mL, respectively; although these concentra- and increasing catabolic activities (McIlwraith tions were not high enough to differ from CON– and Trotter, 1996). Although designed to pro- LPS. At 6 h, the CTM–LRS knee had lower Cu and mote healing, chronic inflammation can lead to Se concentrations (0.33  ± 0.07 μg/mL and 75.6  ± articular degradation (Palmer and Bertone, 1994). 13  ng/mL) than both LPS knees and CON–LRS. Levels of cartilage biomarkers measured in the Concentrations of Cu and Se in CTM–LRS peaked synovial fluid may be influenced by local inflam- at 12 h (0.78 ± 0.07 μg/mL and 147.21 ± 13 ng/mL) matory status. A  useful indicator of inflammation that were similar levels to CTM–LPS 12 h. The ele- and as a marker for the progression of joint dis- vated response to inflammation from either LPS ease is PGE (Bertone et  al., 2001). In the present or the delayed response to repeated arthrocentesis study, PGE concentrations were higher in LPS in- could be due to the form of Cu supplied in the diet jected joints (1210.98 ± 37.43 pg/mL) than in LRS or from the interactions of dietary from of minerals joints (491.11  ± 36.21 pg/mL). Concentrations of supplied with Se. Cobalt was the only mineral dir- PGE peaked at hour 6 in the LPS knee for both ectly affected by diet; horses fed CTM had greater TM sources; however, horses receiving CTM had a mean concentration of Co in the synovial fluid, more pronounced response (3,239 ± 133.86 pg/mL) 8.18 ± 0.5 vs. 5.86 ± 0.5 μg/mL. Therefore, Co glu- vs. CON (2,664  ± 133.91 pg/mL). Similar inflam- coheptonate appears to be more bioavailable than matory responses have been reported in an acute inorganic Co. The 3-way interaction observed in re- LPS model using chickens. When fed Zn amino lation to Co concentrations also conveys that horses acid complex, hens had a greater cytokine produc- fed CTM had a greater Co response to LPS and re- tion (IL-β) 3 h post-challenge before reducing to a peated arthrocentesis than CON horses, exhibiting lower concentration than other diets at 12 h (Cheng higher peak concentrations in the synovial fluid for and Guo, 2004). A  similar increase at 6  h was re- both LPS (hour 6) and LRS (hour 12) knees than ported in yearling and mature horses undergoing CON horses. The exact role of Co in the joint has an intra-articular LPS challenge (de Grauw et al., yet to be elucidated. 2009; Leatherwood et al., 2016). Although reported This study further demonstrates the importance peak values for both studies were higher than peak of the use of controls, as regardless of injection concentration noted in the present study, this could type differed over time. Overall, these data indicate be due to differences in age or to differences in total that acute joint inflammation altered synovial fluid dietary TM fortification. concentrations of TM and that dietary source im- Aggrecan molecules are an essential compo- pacted the resulting degree of response. Currently, nent of the ECM and, due to their highly nega- limited research exits relating TM concentrations tive charge, are responsible for providing the joint and relative time course in equine joints under con- with compressive strength (Frisbie et  al., 2008). trolled inflammation; thus, data reported here are a A  key glycosaminoglycan of aggrecan is chondro- preliminary exploration and do not fully explain the itin sulfate; therefore, the impact of both diet and potential role of complex changes of TMs in joint LPS on aggrecan molecule synthesis was measured inflammation. However, understanding how CTM through the CS846 epitope. A  response of aggre- affect the physiological response of TM concen- can synthesis was exhibited in the presence of LPS trations and the biological roles of TMs within the with highest concentrations at 12  h for CTM and joint further advances our knowledge of the role of 24  h for CON horses. de Grauw et  al. (2009) also TMs in joint health. These data ultimately provide a reported a short-lived inflammation-induced en- foundation for the development of further in-depth hancement of CS846 synthesis, with concentra- studies evaluating specific mechanisms regarding tions highest at 24 h in mature horses. Interestingly, Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. the CTM horses had greater concentrations than CTM horses remained higher at 12 and 24 h before their CON counterparts (245,290  ± 15,663 and decreasing to baseline at 336 h (290 ± 29.85 ng/mL). 196,008  ± 13,653  ng/mL, respectively) in the pre- However, at 336 h, the highest C2C concentrations sent study. In contrast, concentrations in CTM were observed in CTM–CON knees, (340.43  ± horses started to decrease by hour 24; however, 29.85 ng/mL) further exhibiting the need for a sham concentrations returned to baseline by hour 168 for control due to results from repeated arthrocentesis. both TM sources. Multiple enzymes involved in the The rate of recent collagen synthesis can be synthesis of chondroitin sulfate require Mn for syn- measured using CPII (de Grauw et al., 2009). This thesis (Leach, 1971); therefore, a potential explan- molecule is proteolytically cleaved from the pro- ation for the more rapid increase in CTM horses is collagen strand during fibril formation and has a that the complexed Mn may be more readily avail- half-life of 16  h in synovial fluid (Garvican et  al., able for enzyme utilization. 2010). It has also been shown to increase in arth- Inflammation can lead to articular cartilage ritic joints and in osteochondrosis in horses (Frisbie degradation, a key feature in the development of et al., 2008). In addition, CPII concentrations have joint disease. The primary component of articular been shown to increase in response to intra-articular cartilage is type II collagen; its breakdown is highly LPS injection in both growing and mature horses, involved in the development and progression of with variation in concentration between studies (de joint disease (McIlwraith and Trotter, 1996). The Grauw et al., 2009; Lucia et al., 2013; Leatherwood destruction of cartilage results in an accumula- et  al., 2016; Kahn et  al., 2017). Results from the tion of breakdown products in synovial fluid. The present study are consistent with previous work (de analysis of these fragments can help elucidate the Grauw et al., 2009; Lucia et al., 2013; Leatherwood degree of cartilage turnover and potentially high- et al., 2016; Kahn et al., 2017) in that LPS caused light metabolic changes (Garvican et al., 2010). The an increase in CPII concentrations, regardless of breakdown of type II collagen has been measured diet, with highest concentrations at 12 h (1663.09 ± using C2C (de Grauw et al., 2009). The unwinding 175.45  ng/mL) when compared with CON knees or cleavage of collagens by collagenases exposes that peaked later at 24 h (1248.87 ± 175.45 ng/mL). normally hidden epitopes, and these fragments Potential dilution effects of biomarkers could are increased with joint inflammation measured be a confounding factor in synovial fluid biomarker in rabbits, dogs, and horses (Matyas et  al., 2004; analysis; thus, evaluation of ratios looking at the Lucia et al., 2013). Multiple studies have reported anabolic to catabolic processes in the joint may pre- peak concentrations of C2C at 24  h post-LPS in- vent biases (de Grauw et  al., 2011; Te Moller and jection (de Grauw et  al., 2009; Lucia et  al., 2013; van Weeren, 2017). It also allows for observation of Leatherwood et  al., 2016; Kahn et  al., 2017). In metabolic shifts. Previous data have shown a shift the present study, LPS increased C2C concentra- toward synthesis in response to inflammation. This tions, although peak concentrations were exhibited shift allows for damage within the cartilage frame- 6 h post-injection for both CTM and CON (536 ± work to repair; however, replacement of damaged 29.85 and 465 ± 29.85 ng/mL, respectively). matrix may not return the joint to its original state Concentrations of C2C were higher in joints or function (Garvican et  al., 2010). In the present of horses receiving CTM (373.51  ± 9.28  ng/mL) study, the ratio of CPII to C2C was analyzed, and compared with CON (337.36 ± 9.28 ng/mL) horses. even though an increase in C2C was observed in The breakdown and turnover of cartilage collagen CTM horses, the ratio of type II collagen synthesis is largely mediated by matrix metalloproteinases to degradation was unchanged. The intra-articular (MMPs), a family of degradative enzymes that re- LPS tended to increase the ratio due to minimal quire a metal ion for activation (Garvican et  al., increases in anabolic processes, likely a result of 2010). Synthesis of MMPs is regulated by cytokine damage caused by inflammatory mediators. and growth factor production (Milner et al., 2006). In conclusion, the intra-articular LPS chal- As an established model of inflammation, an in- lenge was sufficient in inducing inflammation, creased cytokine production can be expected when cartilage turnover, and aggrecan synthesis, thus al- challenged with LPS. Activation of most MMP re- lowing for the determination of dietary impact on quires Zn; therefore, readily available Zn may be these synovial fluid biomarkers. Compared with allowing for the upregulation of MMPs and the inorganic mineral sources, these data suggest that inflammatory response, resulting in increased con- supplemental intake of a complexed TM source centrations of C2C. Concentrations of C2C for may support ECM turnover in response to an LPS Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses complex on growth performance, carcass characteristics, and challenge as evidenced by an increase in type II col- inflammatory response of beef cattle fed ractopamine hydro- lagen degradation and a more rapid rise in aggre- chloride. J. Anim. Sci. 94:3389–3398. doi:10.2527/jas.2015-0209 can synthesis. Additional research is needed to fully Genther-Schroeder,  O.  N., M.  E.  Branine, and S.  L.  Hansen. understand the impact of TMs and their physio- 2016b. The influence of supplemental Zn-amino acid logical role within the joint and the ability of TM complex and ractopamine hydrochloride feeding duration on growth performance and carcass characteristics of fin- supplementation to delay the onset of joint disease ishing beef cattle. J. Anim. Sci. doi:10.2527/jas.2015-0159 in young horses. Henrotin, Y. E., P. Bruckner, and J. P. Pujol. 2003. The role of reactive oxygen species in homeostasis and degradation of LITERATURE CITED cartilage. Osteoarthr. Cartilage 11:747–755. doi:10.1016/ s1063-4584(03)00150-x AOAC. 2019. Official methods of analysis. 21st ed. Hostetler, C. E., R. L. Kincaid, and M. A. Mirando. 2003. The Gaithersburg, MD: Assoc. Off. Anal. Chem. role of essential trace elements in embryonic and fetal de- Bertone, A. L., J. L. Palmer, and J. Jones. 2001. Synovial fluid velopment in livestock. Vet. J. 166:125–139. doi:10.1016/ cytokines and eicosanoids as markers of joint disease in s1090-0233(02)00310-6 horses. Vet. 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Evaluation of dietary trace mineral supplementation in young horses challenged with intra-articular lipopolysaccharide

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Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Evaluation of dietary trace mineral supplementation in young horses challenged with intra-articular lipopolysaccharide ,2 † Allison A. Millican,* Jessica L. Leatherwood,* Josie A. Coverdale,* Carolyn E. Arnold, ‡ § , # Amanda N. Bradbery,* Connie K. Larson, Emily D. Lamprecht, Sarah H. White,* Chad B. Paulk, Thomas H. Welsh, Jr,* and Tryon A. Wickersham* *Department of Animal Science, Texas A&M University, College Station, TX 77843; Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX 77843; Zinpro Corporation, Eden Prairie, § # MN 55344; Cargill Animal Nutrition, Elk River, MN 55330; and Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506 ABSTRACT:  Sixteen weanling Quarter Horses MIXED procedure of SAS. Results showed a TM (255 ± 22 kg) were utilized in a 56-d trial to evaluate source × LPS × h effect for synovial fluid Co, Cu, the effects of trace mineral (TM) source on intra-ar- and Se (P  <  0.05); concentrations of TM peaked ticular inflammation following a single acute in- at hour 6 and decreased to preinjection values by flammatory insult. Horses were stratified by age, hour 168 in both CON and CTM–LPS knees. A de- sex, and BW and then randomly assigned to dietary layed peak was observed at hour 12 for CTM–LRS. treatment: concentrate formulated with Zn, Mn, Peak synovial fluid Cu and Se concentrations were Cu, and Co as inorganic sources (CON; n = 8) or higher in LPS knees, and Co was highest in CTM– complexed TMs (CTM; n = 8). Added TM were for- LPS. A TM source × h interaction was observed for mulated at iso-levels across treatments and intakes Zn (P < 0.05); concentrations peaked at hour 6 in met or exceeded NRC requirements. Horses were CON vs. hour 12 for CTM. An LPS × h interaction offered 1.75% BW (as-fed) of treatment concentrate was observed for Mn (P < 0.01); synovial concen- and 0.75% BW (as-fed) coastal Bermudagrass hay. tration peaked at hour 6 in LPS knees compared Growth measurements were collected on days 0, 28, with hour 24 in LRS. Synovial PGE , C2C, CPII, and 56, and plasma was collected biweekly for de- and CS846 concentrations were greater with LPS (P termination of Mn, Cu, Zn, and Co concentrations. ≤ 0.01), and C2C was greater (P  <  0.01) in CTM On day 42, carpal joints were randomly assigned compared with CON. Concentrations of CPII and to receive injections of 0.5  ng lipopolysaccharide PGE were unaffected by diet. A TM source × h × (LPS) or sterile lactated Ringer’s solution (LRS; LPS interaction was observed for CS846 (P = 0.02). contralateral control). Synovial fluid was collected Concentrations of CS846 in CTM peaked at 12 h, at preinjection hours (PIH) 0, and 6, 12, 24, 168, whereas CON peaked at a lower concentration at and 336 h post-injection and analyzed for TM con- 24  h (P  <  0.05). Data indicate sufficient intake of centration, prostaglandin E (PGE ), carboxypep- a complexed TM source may support cartilage me- 2 2 tide of type II collagen (CPII), collagenase cleavage tabolism through increased aggrecan synthesis and neopeptide (C2C), and aggrecan chondroitin sulfate type II collagen breakdown following an intra-artic- 846 epitope (CS846). Data were analyzed using the ular LPS challenge in growing horses. Key words: equine, inflammation, lipopolysaccharide, trace minerals 1 2 The authors affirmatively acknowledge that they were Corresponding author: leatherwood@tamu.edu free from influence by Zinpro Corporation and its employ- Received October 3, 2019. ees that would result in any conflict of interest. The authors Accepted January 14, 2020. acknowledge the labor and efforts provided on this project by fellow graduate students: K. Fikes, M. S. Goehring, E. C. Dicksen, C. M. Latham, and C. J. Hartz. 1 Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. © The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non- Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commer- cial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Transl. Anim. Sci. 2020.4:1-16 doi: 10.1093/tas/txaa006 INTRODUCTION MATERIALS AND METHODS Homeostatic maintenance of articulating All care, handling, and procedures for experi- joints requires Cu, Mn, and Zn for turnover of col- ment were approved by the Texas A&M University lagen fibrils and to contribute to molecules within Institutional Animal Care and Use Committee. the extracellular matrix (Hostetler et  al., 2003; Richards et  al., 2010). The role of dietary trace Horses and Treatments minerals (TM) in equine articulating joints remains undetermined; however, information gained from Sixteen weanling Quarter Horses (mean ± other species indicates that metal amino acid-com- SEM; initial BW of 255 ± 22 kg BW; n = 9 colts; plexed TM provide a more biologically available n  =  7 fillies) were used in a complete randomized source of TM (Osorio et  al., 2012). In hens chal- design. Prior to the initiation of dietary treatments, lenged with systemic lipopolysaccharide (LPS) and mares and foals were maintained on the same com- supplemented with Zn amino acid complex, serum mercial concentrate that contained inorganic min- interleukin-1β (IL-1β) concentration increased to eral sources (Producer’s Cooperative Association, 3 h post-induction, but at 12 h, concentrations were Bryan, TX). The concentrate was provided as a lower than hens receiving Zn sulfate (Cheng and creep feed to foals beginning at 90 d of age. Foals Guo, 2004). were weaned at 150 ± 11 d of age and maintained Increased bioavailability and utilization of on the same concentrate until the initiation of the complexed TM may allow for greater incorporation study (233 ± 20 d of age). of TM into articular cartilage and a more rapid Radiographs (lateral, flexed lateral, and crani- achievement in homeostasis following endotoxin al-caudal views) of both radial carpal joints were injection. Utilizing an intra-articular LPS challenge performed at the Texas A&M University Large to induce localized inflammation and cartilage turn- Animal Hospital (College Station, TX) prior to over in young horses provides the ability to evaluate initiation of the study. All horses considered to be the effect of TM source on cartilage metabolism, radiologically normal by a licensed veterinarian joint inflammation, and synovial fluid TM concen- were stratified by age, sex, BW, and BCS, and ran- trations post-induction (Leatherwood et  al., 2016) domly assigned to dietary treatment. Treatment through biomarkers relative to inflammation and diets consisted of isocaloric, isonitrogenous pel- cartilage turnover. leted concentrate formulated with either inorganic Synovial prostaglandin E (PGE ) concen- (CON; 100% inorganic CuSO , ZnSO , and MnSO 2 2 4 4 4 tration increases in response to intra-articular and CoCO ; n = 8) or TM complexes (CTM; zinc LPS. Resulting inflammation influences collagen methionine, manganese methionine, copper lysine, metabolism and aggrecan synthesis by increas- and cobalt glucoheptonate; n = 8). Added levels of ing catabolic collagenase cleavage neopeptide Zn, Mn, Cu, and Co were formulated at iso-levels (C2C), anabolic carboxypropetide of type II col- in both pelleted treatment diets, and daily mineral lagen (CPII), and chondroitin sulfate 846 epitope intakes met or exceeded 2007 NRC minimum re- (CS846; de Grauw et al., 2009; Lucia et al., 2013). commended requirements. All personnel involved Therefore, objectives of this experiment were to in performing the study were blinded to dietary compare effects of dietary TM source (organic treatment. Composited samples of concentrate and vs. inorganic) on growth, joint inflammation, car - hay were analyzed by Equi-Analytical Laboratories tilage metabolism, and synovial fluid TM con- (Ithaca, NY) commercial analysis (Table  1) for centrations in response to an intra-articular LPS dry matter (method 930.15; AOAC, 2019), crude challenge in young horses. petrol (method 990.03; AOAC, 2019), crude fat Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses Table 1. Nutrient composition of concentrates and on days 0, 28, and 56 by the same 3 independent hay (DM basis) fed to weanling horses observers. Rump fat was measured at 5  cm lateral from the midline, halfway between the first coccy- Concentrate geal vertebrae and the ischium (Westervelt et  al., 1 2 3 CON CTM Forage 1976). An altitude stick was used to measure WH Dry matter, % 89.50 91.3 89.16 and HH. Body length and HG were taken using a Nutrient, % DM soft measuring tape. Ultrasonic images were also CP 19.30 19.6 12.51 captured of the longissimus dorsi muscle (LM) were ADF 25.90 23.7 35.51 captured by a certified technician (Designer Genes NDF 40.00 36.1 65.99 Technologies, Inc., Harrison, AR) to determine Fat 7.70 7.9 2.26 Ca 0.94 1.26 0.32 muscle area, back fat thickness (BFT), and intra- P 0.92 1.16 0.29 muscular fat (IMF) deposition. The transducer was Trace minerals , ppm placed to obtain a cross-sectional image taken be- Zn 197.90 184.7 17.9 tween the 13th and 14th as well as the 17th and 18th Cu 51.90 57.9 5.8 ribs. Subcutaneous fat thickness was measured at Mn 216.00 226.9 192.2 three-fourths the distance from the medial end of Co 7.10 9.5 <1.0 the LM; 4 independent images were collected lat- Se 0.73 0.89 erally across the 17th and 18th ribs to estimate IMF Control: pelleted concentrate formulated with 100% inorganic min- within the LM. Four independent images were ne- eral sources (CuSO , ZnSO , and MnSO and CoCO ), supplied for 56 4 4 4 3 cessary to follow Annual Proficiency Testing and d at 1.75% BW (as-fed), n = 8. Certification standard format for data submission. Trace mineral complexes: 7  g of 4-Plex C (Zinpro Corporation, Proper contact between transducer and horse was Eden Prairie, MN) replaced a portion of inorganic added trace min- eral, supplied for 56 d at 1.75% BW (as-fed), n = 8. insured by fitting the transducer with a PIA con- Coastal bermudagrass, Cynodon dactylon, supplied to both treat- tour pad (Animal Ultrasound Services, Ithaca, NY) ment groups at 0.75% BW. designed to conform to the curvature of the horse’s Total mineral content. back. In addition, corn oil was applied to promote Reported on a 100% dry basis. acoustical contact between animal and transducer (Perkins et  al., 1992). An independent laboratory (method 2003.05; AOAC, 2019), acid detergent interpreted all images; personnel were blinded to fiber (ANKOM Technology Method 5), neutral de- treatment (Designer Genes Technologies, Inc.). tergent fiber (ANKOM Technology Method 6), Ca, and P (inductively coupled plasma analysis [ICP]), Incorporation into Plasma and Synovial Fluid and TM concentrations (Fe, Zn, Cu, Mn, Mo, Co, Plasma samples for TM analysis were collected Se) were analyzed by Michigan State University every 14 d into a 6-mL trace element K EDTA, DCPAH (Lansing, MI) with an ICP/mass spec- 10.8  mg, additive tube (BD Vacutainer, Franklin trometer (ICP/MS). Lakes, NJ) prior to the morning feeding. Samples Weanlings received their respective pelleted were immediately placed on ice until centrifugation concentrate at 1.75% BW/d (as-fed) and 0.75% at 2,000 × g for 10 min at 4 °C within 1 h of collec- BW/d (as-fed) of coastal bermudagrass (Cynodon tion. After centrifugation, plasma was aliquoted dactylon) hay divided evenly between 2 feedings at into 1.5-mL microcentrifuge tubes and stored at 0600 and 1800. Horses were fed individually and −80 °C until analysis. A certified veterinarian from maintained in 3 × 3 m stalls with ad libitum access the Texas A&M University Large Animal Clinic to water. Intakes and orts were obtained and meas- (Dr. C. E. Arnold) performed carpal arthrocente- ured daily. Every 7 d, BW was obtained utilizing sis on both radial carpal joints on day 0.  Horses a calibrated digital platform scale (Bastrop Scale were sedated using xylazine HCl, administered Inc., Bastrop, TX) and diets adjusted accordingly. intravenously at recommended dosages. The All horses were allowed 8 h of free exercise in a dry carpal joint was aseptically aspirated using a loca- lot (58.5 × 79.2 m) daily. tion medial to the extensor carpi radialis tendon in the palpable depression between the radial carpal Growth and Performance Characteristics bone and the third carpal bone, to a depth of ap- Body condition score, rump fat (RF), wither proximately 12.7 mm to avoid unnecessary contact height (WH), hip height (HH), body length (BL), with articular cartilage (McIlwraith and Trotter, and heart girth circumference (HG) were taken 1996). Pooled synovial fluid between carpal joints Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. (1 to 4  mL) was transferred into sterile nonaddi- Sample Analysis tive tubes (BD Vacutainer) and was immediately Plasma and synovial fluid samples were sent placed on ice and stored at −80 °C until laboratory to the Michigan State Diagnostics Laboratory analysis. (Lansing, MI) for TM analysis to establish TM composition. In brief, samples were diluted 20-fold Intra-articular LPS Challenge with a solution containing 0.5% EDTA and Triton On day 42 of the study, all horses were sub- X-100, 1% ammonium hydroxide, 2% propanol, jected to an intra-articular LPS challenge. One and 20 ppb of scandium, rhodium, indium, and radiocarpal joint was randomly selected within bismuth as internal standards. The ICP/MS was each horse for injection with LPS, whereas the tuned to yield a minimum of 7,500 cps sensitivity other radiocarpal joint served as a contralateral for 1 ppb yttrium (mass 89), less than 1.0% oxide control (injection of sterile lactated Ringer’s so- level as determined by the 156/140 mass ratio lution; LRS). The use of an LRS joint was based and less than 2.0% double charged ions as deter- on previous data in our laboratory that suggested mined by the 70/140 mass ratio (Wahlen et  al., repeated arthrocentesis influenced local inflam- 2005). Elemental concentrations were calibrated matory status in horses regardless of treatment using a 4-point linear curve of the analyte-inter- (LRS or LPS) as evidenced by an alteration in nal standard response ratio. Standards were from circulating leukocytes, monocytes, or platelets Inorganic Ventures (Christiansburg, VA). In-house (Hunt et al., 2018). serum pools were used as controls. At PIH 0, the carpal joint was aseptically pre- Synovial fluid concentrations CPII, C2C, and pared for arthrocentesis and horses were sedated CS846 were measured using commercially avail- as previously described. Purified LPS derived from able ELISA kits (IBEX Pharmaceuticals Inc., Escherichia coli O55:B5 (Sigma Aldrich, St. Louis, Montreal, QC, Canada) previously validated in MO) was reconstituted and diluted in sterile lac- horses (de Grauw et al., 2009; Lucia et al., 2013). tated Ringer’s solution; individual doses were Synovial fluid samples were analyzed in duplicate, 0.8  mL with a final concentration of 0.5  ng/mL. and standards were prepared according to man- Dosage of LPS was based on previous work in our ufacturer’s recommendations with samples pre- laboratory (Lucia et al., 2013; Leatherwood et al., pared at a 1:4 dilution for both CPII and C2C. 2016; Kahn et al., 2017). The LPS solution was in- Sample dilutions for CS846 ranged from 1:50 serted aseptically into the randomly selected carpal to 1:1,000 depending on time post-injection, to joint, and LRS joints were injected with 0.8  mL remain within detectable limits of the ELISA. of sterile lactated Ringer’s solution after the with- Dilutions were made with calibrator diluents drawal of the PIH 0 sample. Synovial fluid samples provided by the kit prior to beginning the assay. (1 to 4 mL) were obtained at PIH 0 and 6, 12, 24, Mean detectable concentrations for CPII, C2C, 168, and 336 h post-injection). All personnel were and CS846 were 50, 10, and 20  ng/mL, respect- blinded to injection type. ively. Shifts in cartilage metabolism were evaluated All synovial samples were collected and trans- by the ratio of CPII to C2C with individual ratios ferred to sterile nonadditive tubes (BD Vacutainer calculated for each horse. Blood Serum Collection Tubes; Becton-Dickinson Synovial fluid samples were analyzed in du- and Company, Franklin Lakes, NJ) and imme- plicate for concentrations of PGE utilizing an diately placed on ice until aliquoted into 1.5-mL enzyme-linked immunoassay (R&D Systems, microcentrifuge tubes. Aliquots were stored at Minneapolis, MN), previously validated in horses −80  °C until later analysis of C2C, CPII, CS846, (Bertone et al., 2001; de Grauw et al., 2006; Lucia PGE , and TM concentrations. All horses were et  al., 2013). Samples intended for PGE analysis 2 2 monitored for signs of anaphylaxis over the initial were diluted from 1:1 to 1:10 depending on time 24  h post-injection. Rectal temperature (RT; °C), post-injection to remain within detectable limits of heart rate (HR; beats/min), and respiratory rate the ELISA; dilutions were prepared using the cali- (RR; breaths/min) were recorded prior to arthro- brator diluent provided by the kit with a mean de- centesis at PIH 0 and at 6, 12, and 24 h post-injec- tectable dose of PGE of 39 pg/mL. tion. Carpal circumference (cm) was measured at Final concentrations of all markers were read the level of the accessory carpal bone with a soft using a microplate reader (Synergy H1, Biotek, tape measure that was performed by a single indi- Winooski, VT) with optical density set at 450 nm. vidual to maintain consistency. Intracoefficients of variation for CPII, C2C, and Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses PGE were less than or equal to 15% and less than evaluate normal distributions for traits based on 20% for CS846. continuous variables. One biomarker (CPII) exhib- ited non-normal data; therefore, the data were log transformed for normalization. Outliers were iden- Statistical Analysis tified using box plots of the residuals and removed Intake data were analyzed by using the MIXED if greater than 3 SD from the mean. Data are re- procedure of SAS v9.4 (SAS Inst. Inc., Cary, NC). ported as mean ± SEM. The model contained fixed effects of TM source and time (d). Initial, final, and delta values for growth RESULTS parameters were analyzed using the MIXED pro- cedure of SAS v9.4 (SAS Inst. Inc., Cary, NC). The Target intakes of 1.75% BW (as-fed) from con- model contained a fixed effect of TM source and centrate and 0.75% BW (as-fed) from hay were used a random variable of horse within treatment. achieved as horses consumed their respective diets Plasma mineral concentration utilized the same with no significant refusals across treatment groups model with an additional fixed effect of time (d) over the 56-d trial. Weanling horses consumed en- and a TM source × d interaction. ergy and associated nutrients to meet or exceed re- In response to the LPS challenge, statistical commended requirements (NRC, 2007). Total dry analysis of synovial fluid biomarker and TM con- matter intake throughout the 56-d study did not centration were analyzed using PROC MIXED of differ between TM source (P = 0.89), with daily in- SAS v9.4 (SAS Inst. Inc., Cary, NC). The model takes of 5.74 and 5.71 ± 0.15 kg for CON and CTM, contained fixed effects of TM source, time (h), respectively. Individually, concentrate and forage injection type (LPS), and their respective inter- intakes were not different between TM sources actions. This model included a random effect of (P = 0.54 and P = 0.47, respectively). Similarly, TM horse within treatment and a repeated variable source did not affect (P = 0.78) final BW (285.8 and (time). The covariate structure was utilized to 288.8 ± 5.57 kg for CON and CTM, respectively), specify a random effect for differences between with all horses gaining 32  ± 2  kg during the 56 d animals within treatment, creating a correlation (P = 0.90), validating the diets were isocaloric. structure within animals that decreases with the No effect of TM source was observed in syn- increasing amount of time between measurements ovial biomarker concentrations during the pre-LPS (Littell et  al., 1998). The model for synovial fluid dietary adaptation period; however, a TM source TM analysis also included hour 0 as a covariate. × d interaction was observed for CS846 (P = 0.05; Post hoc comparison of TM source and individual Table  2). Synovial fluid CS846 concentration in- time points was conducted using a paired t-test. creased from days 0 to 42 in CTM horses, while Significant differences were declared as P ≤ 0.05, remaining similar from day 0 to day 42 in CON and P ≤ 0.10 was considered a trend toward sig- horses. Conversely, the ratio of CPII to C2C in- nificance. Plots of residual variation were used to creased in all horses from days 0 to 42 (P  =  0.04), Table 2.  Cartilage biomarkers and inflammatory markers within the synovial fluid of weanling Quarter Horses before (day 0) and (day 42) 42 d of receiving a pelleted concentrate at 1.75% BW (as-fed) containing either inorganic (CON; n = 8) or organic (CTM; n = 8) dietary trace mineral supplementation Dietary treatments CON CTM P-value Synovial fluid variable Day 0 Day 42 Day 0 Day 42 SEM TM source d TM source × d CPII, ng/mL 764.65 967.17 713.96 988.23 231.33 0.96 0.13 0.82 C2C, ng/mL 270.93 260.86 276.66 297.89 29.23 0.48 0.80 0.49 CPII:C2C 2.76 3.82 2.54 3.90 0.91 0.95 0.04 0.80 a a a b CS846, ng/mL 23,820 24,803 19,701 29,717 3,411 0.92 0.02 0.05 PGE , pg/mL 596.85 336.37 750.68 423.02 87.90 0.13 <0.01 0.64 Control (CON): pelleted concentrate formulated with 100% inorganic mineral sources (CuSO , ZnSO , and MnSO and CoCO ), supplied at 4 4 4 3 1.75% BW (as-fed), n = 8. Trace mineral complexes (CTM): 7 g of 4-Plex C (Zinpro Corporation, Eden Prairie, MN) replaced a portion of inor- ganic added trace mineral, supplied at 1.75% BW (as-fed), n = 8. 2 ; ; CPII = carboxypropetide of type II collagen; C2C = collagenase cleavage neopeptide CS846 = chondroitin sulfate 846 epitope PGE  = pros- taglandin E . ab Within row, means with different letters differ (P < 0.05). Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. whereas PGE decreased over time (P  <  0.01), re- Table 3.  Growth parameters and composition of gardless of TM source (Table 2). weanling Quarter Horses before (day 0)  and after 56 d of receiving a pelleted concentrate at 1.75% BW (as-fed) containing either inorganic (CON; Growth Metrics n = 8) or organic (CTM; n = 8) dietary trace min- eral supplementation Growth measurements including WH, HH, HG, and BL were not affected (P ≥ 0.56) by TM Dietary source (Table  3). Similarly, no differences between treatments TM sources were observed for final IMF, BFT, or 1 2 Variable CON CTM SEM P-value rump fat (P > 0.10). However, final BCS tended to BW, kg be lower (P = 0.06) for horses fed CTM when com- Day 0 256.4 253.7 8.0 0.82 Day 56 288.8 285.8 7.6 0.78 pared with CON. Final LM area at both measured Wither height, cm locations was not affected by TM source (P ≥ 0.60). Day 0 128.19 127.56 0.97 0.65 Overall change in area at the 17th and 18th rib was Day 56 131.68 131.60 0.67 0.93 unaffected by TM source, but a positive change Hip height, cm in area over the 56-d trial was observed in CON Day 0 134.54 134.38 1.12 0.92 2 2 (+1.72 cm ) and CTM treatments (+1.97 cm ), indi- Day 56 137.80 138.19 0.99 0.78 cating growth. Heart girth, cm Day 0 143.25 141.67 1.97 0.56 Plasma Mineral Concentration Day 56 149.52 148.13 1.82 0.60 Body length, cm No significant interaction or main effects of Day 0 135.21 134.94 1.39 0.89 TM source and d were observed for Mn (P ≥ 0.18) Day 56 144.74 145.31 1.35 0.77 with average concentrations of 1.73 and 1.62  ± Body condition score Day 0 5.65 5.52 0.16 0.58 0.09  ng/mL for CTM and CON, respectively. No Day 56 6.55 6.08 0.16 0.06 interaction or effect of TM was observed for Cu Rump fat (P ≥ 0.41); however, an effect of day (P < 0.01) was Day 0 0.14 0.15 0.006 0.79 present. Plasma Cu concentrations increased from Day 56 0.16 0.16 0.005 0.86 days 0 to 42 (P < 0.01; 1.07 to 1.22 ± 0.05 μg/mL for Intramuscular fat, % days 0 and 42, respectively) and remained elevated Day 0 3.62 3.65 0.17 0.91 through day 56. A TM source × d interaction was Day 56 3.64 3.48 0.17 0.52 present for Zn (P  =  0.02; Fig.  1). Concentrations Back fat thickness, 13th and 14th rib decreased in CON from days 0 to 56 while increas- Day 0 0.17 0.15 0.02 0.42 ing in CTM from day 14 to day 28 before declining Day 56 0.16 0.17 0.01 0.57 to levels similar to CON by day 42. A  significant Back fat thickness, 17th and 18th rib TM source × d interaction was observed for Co Day 0 0.14 0.14 0.01 0.74 Day 56 0.14 0.14 0.01 0.81 concentrations (P  <  0.01; Fig.  2). Cobalt concen- LM area, cm , 13th and 14th rib trations increased from days 0 to 14 for all horses Day 0 10.57 10.27 0.45 0.65 regardless of TM source; however, horses receiving Day 56 11.75 12.00 0.59 0.77 CTM had greater Co plasma concentrations com- LM area, cm , 17th and 18th rib pared with the control CON, beginning at day 14 Day 0 12.01 11.63 0.52 0.61 through day 56 of the study. Day 56 13.73 13.60 0.28 0.60 Control: pelleted concentrate formulated with 100% inorganic min- LPS Challenge eral sources (CuSO , ZnSO , and MnSO and CoCO ), supplied for 56 4 4 4 3 d at 1.75% BW (as-fed), n = 8. Clinical assessment  During the LPS chal- Trace mineral complexes: 7  g of 4-Plex C (Zinpro Corporation, lenge, no TM source × h interactions were present Eden Prairie, MN) replaced a portion of inorganic added trace min- for clinical parameters, including HR, RR, and eral, supplied for 56 d at 1.75% BW (as-fed), n = 8. Rump fat thickness measured by ultrasound. RT (P ≥ 0.33; data not shown). Average beats per minute were 48.4 and 50.9  ± 21.6 (P ≥ 0.22), for CON and CTM horses, respectively. Respiration PIH 0 levels at hour 12 (16  ± 2 breaths/min) and rate was affected by hour (P  <  0.01), with an in- hour 24 (15  ± 2 breaths/min). An effect of h was crease from PIH 0 (20 ± 2 breaths/min) to hour 6 also observed for RT (P  =  0.01). Rectal tempera- (24  ± 2 breaths/min), before decreasing to below ture increased from PIH 0 (38.0 ± 0.1 °C) to 38.4 ± Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses Figure 1. Mean plasma Zn concentrations over 56-d trial in weanling horses receiving a pelleted concentrate at 1.75% BW (as-fed) containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc methionine, manganese methionine, 4 4 4 3 copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Trace mineral source × d interaction (P < 0.05). *CTM greater than CON at day 28 (P < 0.05). Figure 2. Mean plasma Co concentrations over 56-d trial in weanling horses receiving a pelleted concentrate fed at 1.75% BW (as-fed) con- taining either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes Zn methionine, Mn methionine, 4 4 4 3 Cu lysine, and Co glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Trace mineral source × d interaction (P < 0.01). *CTM higher than CON (P < 0.01). 0.1  °C at hour 6 before returning to baseline by addition, at hour 12, Co was greater in CTM–CON hour 24 (38.0 ± 0.1 °C). All values remained within than CON–LPS and CON–LRS (P < 0.01). normal physiological limits throughout the 24-h A TM source × LPS × h effect was observed period. An LPS × h interaction was observed for for Cu (P < 0.01; Fig. 4). Concentrations of Cu in- carpal circumference (P < 0.01), as the LPS injected creased in both LPS and CON–LRS to peak con- knees increased to hour 12 and remained elevated centrations by hour 6 then decreased to hour 168, to hour 24, whereas the LRS knee increased in cir- whereas concentration of Cu was lowest at hour 6 cumference to hour 12 and returned to baseline by in CTM–LRS (P  =  0.01). Concentrations of Cu hour 24. Carpal circumference increased over time in CTM–LRS increased from hours 6 to 12 and (P < 0.01; data not shown), but was not affected by then declined to hour 168. Peak concentration for TM source. CTM–LRS was observed at hour 12, and the Cu Synovial fluid mineral concentration  A TM concentration was similar to other knees at that source × LPS × h interaction was observed for Co time point. No differences were observed at hour (P  <  0.01; Fig.  3). Cobalt concentration at hour 168 or 336. Trace mineral source × LPS × h effect 6 was the greatest in CTM–LPS (P  <  0.01). In was also observed for Se (P  =  0.02; Fig.  5), with Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. Figure 3. Mean synovial fluid cobalt concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc methionine, 4 4 4 3 manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pel- leted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Cobalt concentrations: trace mineral source × LPS abc × h interaction (P < 0.01). Denotes differences within each time point between trace mineral source and LPS (P < 0.05). Figure 4. Mean synovial fluid copper concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc methionine, 4 4 4 3 manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pel- leted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Copper concentrations: trace mineral source × LPS abc × h interaction (P = 0.01). Differences within each time point between trace mineral source and LPS (P < 0.05). concentrations following the same basic pattern CON and hour 12 for CTM before decreasing to as Cu. Concentrations of Se were lowest in CTM– hour 168. At hours 12, 24, and 168, CON had a LRS at hour 6 vs. other knees (P < 0.01) and then lower concentration of Zn than CTM (P  <  0.01). increased to similar concentrations at hour 12 to An LPS × h interaction (P  <  0.01) was observed those in other knees. Concentrations of Se then for Zn; concentrations were higher in LPS knees at declined in all knees to hour 168, and maintained hours 6 and 24 (P ≤ 0.01) and tended to be higher at similar concentrations to hour 336. No TM source hour 12 than CON (P = 0.06). × LPS × h interaction was present for Zn (P = 0.21; No TM source × h × LPS interaction (P = 0.74; Fig. 6), although concentrations followed the same Fig. 7) was observed for Mn; however, an h × LPS general pattern as Cu and Se. A  TM source × h interaction was present (P < 0.01). Manganese con- interaction was observed for Zn (P  =  0.02). Zinc centration increased in both LPS knees to hour 6 increased in concentration, peaking at hour 6 in and then decreased steadily to baseline levels by Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses Figure 5. Mean synovial fluid selenium concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc methionine, 4 4 4 3 manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Selenium concentrations: trace mineral source × abc LPS × h interaction (P = 0.02). Differences within each time point between trace mineral source and LPS (P < 0.05). Figure 6. Mean synovial fluid zinc concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc methionine, 4 4 4 3 manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pel- leted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Zinc concentrations: trace mineral source × LPS × h (P = 0.21), trace mineral source × h (P < 0.05), h × LPS (P < 0.01). *Differences within each time point between trace mineral source (P < 0.05). Differences within each time point between injection type (LPS). hour 168. LRS knees displayed an increase from Cartilage markers  An LPS × h inter- hour 12 to 24 resulting in higher Mn concentra- action was present for synovial C2C (P  <  0.01; tion in LRS compared with LPS knees at hour 24 Fig.  9). Concentrations of C2C in LPS knees (P < 0.01). Concentration of Mn in LRS knees then were greater than concentrations in LRS knees decreased to baseline levels by hour 168. at hours 6, 12, 24, and 168 (P ≤ 0.04). A  ten- Synovial inflammation  An LPS × h interaction dency for an interaction of TM source × LPS (P ≤ 0.01; Fig. 8) was observed for synovial PGE . (P  =  0.09) was observed with concentrations Concentration of PGE was greater in the LPS in- of C2C being greater in CTM–LPS compared jected knee when compared with LRS at hours 6, with CON–LPS (P  <  0.01). An LPS × h inter- 12, and 24 (P  <  0.01). Synovial PGE was not af- action (P ≤ 0.01; Fig.  10) was observed for fected by TM source (P = 0.13). anabolic CPII with LPS injection resulting in Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. Figure 7. Mean synovial fluid manganese concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments consisted of pel- leted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes zinc me- 4 4 4 3 thionine, manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Manganese concentrations: trace mineral source × h × LPS interaction (P = 0.74), LPS × h interaction (P < 0.01), trace mineral source (P = 0.35). *Differences between LRS knees and LPS knees (P < 0.01). Figure 8. Mean synovial prostaglandin E (PGE ) concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from 2 2 Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treatments con- sisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral complexes 4 4 4 3 zinc methionine, manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Trace mineral source × LPS × h (P = 0.48), LPS × h interaction (P < 0.01), trace mineral source (P = 0.13). greater CPII concentrations at 6, 12, and 168  h in LPS knees than LRS knees with no difference (P ≤ 0.01) compared with LRS. The CPII to C2C between dietary mineral sources. At hour 12, knees ratio was not significantly affected by TM source injected with LPS in CTM horses had the greatest (P  =  0.57), LPS (P  =  0.11), or hour (P  =  0.19; concentration of CS846 (P < 0.01) followed by LPS data not shown). knees in CON horses; LRS knees remained near A significant interaction of TM source × LPS baseline. By hour 24, LPS knees in CON horses × h (P  =  0.02; Fig.  11) was observed for synovial had higher CS846 concentrations than CTM horses CS846 concentrations. Prior to injection of LPS and LRS knees had increased above baseline but (PIH 0)  there were no differences in CS846 levels; remained lower than LPS. By hour 168, no differ- however, 6 h after LPS injection, CS846 was higher ences were detected. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses Figure 9. Mean synovial collagenase cleavage neopeptide (C2C) concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treat- ments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral 4 4 4 3 complexes zinc methionine, manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Trace mineral source × LPS × h (P = 0.35), LPS × h (P < 0.01), trace mineral source × LPS (P = 0.09). Figure 10. Mean synovial carboxypeptide of type II collagen (CPII) concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treat- ments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral 4 4 4 3 complexes zinc methionine, manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Trace mineral source × LPS × h (P = 0.99), LPS × h (P < 0.01), trace mineral source (P = 0.82). DISCUSSION complex has been shown to increase long bone length and width in embryos and chicks (Favero The present study examined the effect of com- et al., 2013). Limited data exist observing the effect plexed Zn, Cu, Mn, and Co source on growth and of complexed TMs on equine growth; however, intra-articular inflammation in growing horses. differences between TM proteinates and inorganic Our study did not detect a positive effect of CTM sources were evaluated in yearling horses fed for on growth of yearling horses, which is in contrast 112 d (Ott and Johnson, 2001). No effect of source with the prior reports that complexed TM in- on BW, WH, HG, or BL gains was reported; how- creased growth in beef cattle and calves (Osorio ever, HH gain was greater for horses receiving pro- et  al., 2012; Genther-Schroeder et  al., 2016a, b). tienates than the inorganic supplemented horses. Osorio et  al. (2012) reported that wither height In the current 56-d study, all horses, regardless was increased in calves fed complexed TMs (Zn, of diet, increased in BW, HH, WH, BL, and HG Mn, Cu, and Co) vs. inorganic sources at weaning. circumference. In addition, the addition of dietary Zn amino acid Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. Figure 11. Mean synovial chondroitin sulfate epitope 846 (CS846) concentrations following a 0.5-ng intra-articular lipopolysaccharide (LPS; derived from Escherichia coli O55:B5) injection at 0 to 336 h post-injection or lactated Ringer’s solution (LRS; contralateral control). Dietary treat- ments consisted of pelleted concentrates containing either 100% inorganic ZnSO , CuSO , and MnSO and CoCO (CON; n = 8) or trace mineral 4 4 4 3 complexes zinc methionine, manganese methionine, copper lysine, and cobalt glucoheptonate (CTM; n = 8). Added levels of Zn, Mn, Cu, and Co were at iso-levels in both pelleted treatment diets, and daily mineral intakes met or exceeded NRC minimum requirements. Significant interactions: abc TM source × LPS × h interaction (P < 0.02). Differences in concentration among TM sources at specific time points post-intra-articular injection (P < 0.05). In previous studies exposing young horses to of each mineral increased to 6 h before returning to intra-articular LPS, clinical responses for HR, RR, baseline or below baseline levels by 168 h post-injec- and RT showed no signs of systemic illness (de tion in all knees except CTM–LRS, which showed a Grauw et al., 2009; Lucia et al., 2013). In the pre- delayed increase at 12 h. sent study, clinical measures also remained within In human osteoarthritic patients, similar in- normal physiological ranges for horses of this age creases in synovial fluid Cu and positive correl- and demonstrated the inflammatory response re- ations between synovial fluid Zn and Cu have been mained localized. Joint circumference increased reported in response to inflammation (Yazar et al., regardless of TM source or intra-articular treat- 2005). The increases in concentrations of Zn, Cu, ment (LPS vs. LRS) above baseline values at 6  h, and Se in response to LPS could be due to their increased at 12  h, and began decreasing at 24  h; role in combating free radical formation. The pres- however, values did not return to baseline by 336 h. ence of reactive oxygen species is damaging to The lack of differences between joints receiving the extracellular matric (ECM) both structurally LPS vs. LRS further validates the inclusion of and functionally (Henrotin et  al., 2003). Together, an LRS sham-injected knee in the LPS model to Se-containing glutathione peroxidase and super- account for effects of repeated arthrocentesis. Data oxide dismutase (SOD) are the major antioxidant collected in the present study agree with previous defense systems against oxygen free radicals. Three literature, indicating 0.5 ng LPS causes acute syno- isoforms of SOD exist in mammals: cytoplasmic vitis resulting in minor carpal circumference in- Cu/Zn SOD, mitochondrial Mn SOD, and extracel- creases with minimal physiological changes of HR, lular Cu/Zn SOD (Fukai and Ushio-Fukai, 2011). RR, and RT (Lucia et al., 2013; Kahn et al., 2017). Decreased SOD activity is exhibited in humans and Synovial fluid TM concentrations in response mice with osteoarthritis (Regan et  al., 2005). The to an inflammatory insult have not previously been increase in Cu could potentially be due to an in- reported in horses. Due to roles for various TMs as crease in the Cu-containing acute phase protein, enzyme activators and cofactors, changes in concen- ceruloplasmin, which appears to exert antioxidant trations have potential to affect other biochemical effects in human knee-joint synovial fluid (Blake indicators. In the present study, all TMs measured et al., 1981). varied over time, likely a result of inflammation as- In contrast to Zn, Cu, and Se, synovial fluid sociated with both LPS and repeated arthrocentesis. concentration of Mn was minimally responsive Trace minerals, Zn, Cu, Mn, and Se, were directly to LPS. Specifically, manganese concentrations of influenced by LPS injection, increasing in the syn- 6.41 and 4.81  ng/mL were exhibited in the CTM ovial fluid post-injection. Concentrations of Cu, and CON–LRS knees, respectively, at 24  h. These Zn, and Se responded similarly as concentrations values are approximately 7- to 10-fold higher than Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Dietary metal complexes in young horses values at hour 0, whereas Mn values in LPS knees interaction networks of TM under inflammatory were only 2 to 2.5 times greater (CTM and CON, conditions within the joint. Furthermore, research respectively) than hour 0. The decrease in Mn in the identifying the source of increased TM in equine LPS knees could be attributed to increased chon- synovial fluid would provide improved under - droitin sulfate synthesis, supported by the increase standing of how nutritional mineral status and car- in CS846 in response to the LPS. tilage health could result in mineral extraction from The 3-way interactions observed between diet, articular cartilage. h, and LPS for Cu and Se indicate dietary source af- Articular cartilage integrity is heavily dependent fected degree of response between knees. The peak on the balance of metabolic activities (Mueller and concentrations were highest in the CTM–LPS knee Tuan, 2011). The presence of inflammation causes for Cu and Se, 0.99  ± 0.07  μg/mL and 228.62  ± altered cartilage metabolism by decreasing anabolic 14.2 ng/mL, respectively; although these concentra- and increasing catabolic activities (McIlwraith tions were not high enough to differ from CON– and Trotter, 1996). Although designed to pro- LPS. At 6 h, the CTM–LRS knee had lower Cu and mote healing, chronic inflammation can lead to Se concentrations (0.33  ± 0.07 μg/mL and 75.6  ± articular degradation (Palmer and Bertone, 1994). 13  ng/mL) than both LPS knees and CON–LRS. Levels of cartilage biomarkers measured in the Concentrations of Cu and Se in CTM–LRS peaked synovial fluid may be influenced by local inflam- at 12 h (0.78 ± 0.07 μg/mL and 147.21 ± 13 ng/mL) matory status. A  useful indicator of inflammation that were similar levels to CTM–LPS 12 h. The ele- and as a marker for the progression of joint dis- vated response to inflammation from either LPS ease is PGE (Bertone et  al., 2001). In the present or the delayed response to repeated arthrocentesis study, PGE concentrations were higher in LPS in- could be due to the form of Cu supplied in the diet jected joints (1210.98 ± 37.43 pg/mL) than in LRS or from the interactions of dietary from of minerals joints (491.11  ± 36.21 pg/mL). Concentrations of supplied with Se. Cobalt was the only mineral dir- PGE peaked at hour 6 in the LPS knee for both ectly affected by diet; horses fed CTM had greater TM sources; however, horses receiving CTM had a mean concentration of Co in the synovial fluid, more pronounced response (3,239 ± 133.86 pg/mL) 8.18 ± 0.5 vs. 5.86 ± 0.5 μg/mL. Therefore, Co glu- vs. CON (2,664  ± 133.91 pg/mL). Similar inflam- coheptonate appears to be more bioavailable than matory responses have been reported in an acute inorganic Co. The 3-way interaction observed in re- LPS model using chickens. When fed Zn amino lation to Co concentrations also conveys that horses acid complex, hens had a greater cytokine produc- fed CTM had a greater Co response to LPS and re- tion (IL-β) 3 h post-challenge before reducing to a peated arthrocentesis than CON horses, exhibiting lower concentration than other diets at 12 h (Cheng higher peak concentrations in the synovial fluid for and Guo, 2004). A  similar increase at 6  h was re- both LPS (hour 6) and LRS (hour 12) knees than ported in yearling and mature horses undergoing CON horses. The exact role of Co in the joint has an intra-articular LPS challenge (de Grauw et al., yet to be elucidated. 2009; Leatherwood et al., 2016). Although reported This study further demonstrates the importance peak values for both studies were higher than peak of the use of controls, as regardless of injection concentration noted in the present study, this could type differed over time. Overall, these data indicate be due to differences in age or to differences in total that acute joint inflammation altered synovial fluid dietary TM fortification. concentrations of TM and that dietary source im- Aggrecan molecules are an essential compo- pacted the resulting degree of response. Currently, nent of the ECM and, due to their highly nega- limited research exits relating TM concentrations tive charge, are responsible for providing the joint and relative time course in equine joints under con- with compressive strength (Frisbie et  al., 2008). trolled inflammation; thus, data reported here are a A  key glycosaminoglycan of aggrecan is chondro- preliminary exploration and do not fully explain the itin sulfate; therefore, the impact of both diet and potential role of complex changes of TMs in joint LPS on aggrecan molecule synthesis was measured inflammation. However, understanding how CTM through the CS846 epitope. A  response of aggre- affect the physiological response of TM concen- can synthesis was exhibited in the presence of LPS trations and the biological roles of TMs within the with highest concentrations at 12  h for CTM and joint further advances our knowledge of the role of 24  h for CON horses. de Grauw et  al. (2009) also TMs in joint health. These data ultimately provide a reported a short-lived inflammation-induced en- foundation for the development of further in-depth hancement of CS846 synthesis, with concentra- studies evaluating specific mechanisms regarding tions highest at 24 h in mature horses. Interestingly, Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/4/2/txaa006/5707709 by guest on 18 February 2020 Millican et al. the CTM horses had greater concentrations than CTM horses remained higher at 12 and 24 h before their CON counterparts (245,290  ± 15,663 and decreasing to baseline at 336 h (290 ± 29.85 ng/mL). 196,008  ± 13,653  ng/mL, respectively) in the pre- However, at 336 h, the highest C2C concentrations sent study. In contrast, concentrations in CTM were observed in CTM–CON knees, (340.43  ± horses started to decrease by hour 24; however, 29.85 ng/mL) further exhibiting the need for a sham concentrations returned to baseline by hour 168 for control due to results from repeated arthrocentesis. both TM sources. Multiple enzymes involved in the The rate of recent collagen synthesis can be synthesis of chondroitin sulfate require Mn for syn- measured using CPII (de Grauw et al., 2009). This thesis (Leach, 1971); therefore, a potential explan- molecule is proteolytically cleaved from the pro- ation for the more rapid increase in CTM horses is collagen strand during fibril formation and has a that the complexed Mn may be more readily avail- half-life of 16  h in synovial fluid (Garvican et  al., able for enzyme utilization. 2010). It has also been shown to increase in arth- Inflammation can lead to articular cartilage ritic joints and in osteochondrosis in horses (Frisbie degradation, a key feature in the development of et al., 2008). In addition, CPII concentrations have joint disease. The primary component of articular been shown to increase in response to intra-articular cartilage is type II collagen; its breakdown is highly LPS injection in both growing and mature horses, involved in the development and progression of with variation in concentration between studies (de joint disease (McIlwraith and Trotter, 1996). The Grauw et al., 2009; Lucia et al., 2013; Leatherwood destruction of cartilage results in an accumula- et  al., 2016; Kahn et  al., 2017). Results from the tion of breakdown products in synovial fluid. The present study are consistent with previous work (de analysis of these fragments can help elucidate the Grauw et al., 2009; Lucia et al., 2013; Leatherwood degree of cartilage turnover and potentially high- et al., 2016; Kahn et al., 2017) in that LPS caused light metabolic changes (Garvican et al., 2010). The an increase in CPII concentrations, regardless of breakdown of type II collagen has been measured diet, with highest concentrations at 12 h (1663.09 ± using C2C (de Grauw et al., 2009). The unwinding 175.45  ng/mL) when compared with CON knees or cleavage of collagens by collagenases exposes that peaked later at 24 h (1248.87 ± 175.45 ng/mL). normally hidden epitopes, and these fragments Potential dilution effects of biomarkers could are increased with joint inflammation measured be a confounding factor in synovial fluid biomarker in rabbits, dogs, and horses (Matyas et  al., 2004; analysis; thus, evaluation of ratios looking at the Lucia et al., 2013). Multiple studies have reported anabolic to catabolic processes in the joint may pre- peak concentrations of C2C at 24  h post-LPS in- vent biases (de Grauw et  al., 2011; Te Moller and jection (de Grauw et  al., 2009; Lucia et  al., 2013; van Weeren, 2017). It also allows for observation of Leatherwood et  al., 2016; Kahn et  al., 2017). In metabolic shifts. Previous data have shown a shift the present study, LPS increased C2C concentra- toward synthesis in response to inflammation. This tions, although peak concentrations were exhibited shift allows for damage within the cartilage frame- 6 h post-injection for both CTM and CON (536 ± work to repair; however, replacement of damaged 29.85 and 465 ± 29.85 ng/mL, respectively). matrix may not return the joint to its original state Concentrations of C2C were higher in joints or function (Garvican et  al., 2010). In the present of horses receiving CTM (373.51  ± 9.28  ng/mL) study, the ratio of CPII to C2C was analyzed, and compared with CON (337.36 ± 9.28 ng/mL) horses. even though an increase in C2C was observed in The breakdown and turnover of cartilage collagen CTM horses, the ratio of type II collagen synthesis is largely mediated by matrix metalloproteinases to degradation was unchanged. The intra-articular (MMPs), a family of degradative enzymes that re- LPS tended to increase the ratio due to minimal quire a metal ion for activation (Garvican et  al., increases in anabolic processes, likely a result of 2010). Synthesis of MMPs is regulated by cytokine damage caused by inflammatory mediators. and growth factor production (Milner et al., 2006). In conclusion, the intra-articular LPS chal- As an established model of inflammation, an in- lenge was sufficient in inducing inflammation, creased cytokine production can be expected when cartilage turnover, and aggrecan synthesis, thus al- challenged with LPS. Activation of most MMP re- lowing for the determination of dietary impact on quires Zn; therefore, readily available Zn may be these synovial fluid biomarkers. Compared with allowing for the upregulation of MMPs and the inorganic mineral sources, these data suggest that inflammatory response, resulting in increased con- supplemental intake of a complexed TM source centrations of C2C. 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