Quarter Horse Yearlings Research Articles

86 Evaluation of dietary Saccharomyces cerevisiae fermentation product on markers of joint inflammation in young, exercising horses following an intra-articular lipopolysaccharide challenge

Abstract Including Saccharomyces cerevisiae fermentation product (SCFP) into the diets of young, exercising horses may prolong their performance career by optimizing the intra-articular environment. Our objective was to evaluate the effect of dietary SCFP on joint inflammation in yearlings undergoing regular exercise, challenged with intra-articular lipopolysaccharide (LPS). Thirty Quarter Horse yearlings (374 ± 25 kg BW; 562 ± 16 d of age; 15 fillies and 15 geldings) were used in a 60-d study to test the hypothesis that dietary SCFP (TruEquine C, Diamond V Mills, Inc.) ameliorates joint inflammation following an acute inflammatory insult. Horses were stratified by BW, age, sex, and randomly assigned to dietary treatments (n = 10/treatment): Control (0), 46, or 92 mg/kg BW SCFP. Treatments were top-dressed onto a custom-formulated concentrate void of added microbials offered at 1% BW/d (as-fed) every 12 h. Horses were individually stalled (3.6 m × 7.3 m) and offered ad libitum Coastal bermudagrass hay. Using a free-stall exerciser, horses were exercised following a progressive workload regimen for 30 min/d, 5 d/wk. On d 46, all horses underwent an intra-articular LPS challenge where each horse had one radial carpal joint randomly assigned to receive either 0.8 mL of a 0.5 ng LPS solution or sterile lactated Ringer’s solution (LRS) as a contralateral control. Synovial fluid samples were obtained pre-injection (h 0) and at 6, 12, 24, and 336 h post-injection and analyzed for prostaglandin E2 (PGE2), a pro-inflammatory prostaglandin via commercial ELISA, and for chemokine-concentration (signaling proteins that direct immune cells; CCL2, and CCL11) and cytokines (inflammatory mediators; TNFα and IL-10) using a multiplex platform via commercial laboratory. Non-normal data (PGE2, IL-10, and CCL2) were log transformed, and all markers were analyzed using MIXED procedure of SAS v9.4. Mean separation was achieved with orthogonal contrasts, and contralateral control carpi were used as a covariate across all hours. There was no effect of SCFP on synovial logPGE2 (P = 0.42), logCCL2 (P = 0.90), CCL11 (P = 0.26), or logIL-10 (P = 0.23). However, there was a treatment × hour interaction for CCL11 (P = 0.04) where the Control had increased concentrations compared with SCFP treatment groups at 6 h post-injection. Furthermore, logIL-10 also had a treatment × hour interaction (P = 0.05) where the 46mg/kg BW group had decreased concentrations at h 12 compared with the Control and 92mg/kg BW group. The main effect of treatment for TNFα (P = 0.04) revealed that the 92mg/kg BW group had less concentrations than the 46mg/kg BW group and tended to have less concentrations than the Control group across all hours. Results indicate that SCFP may help mitigate inflammation markers following an acute intra-articular inflammatory insult.

83 Effect of a Saccharomyces cerevisiae fermentation product on intake and digestion in young horses undergoing exercise training

Young growing horses subjected to exercise have greater dietary requirements than horses at maintenance necessitating the delivery of a more nutrient dense diet. Inclusion of a grain concentrate in the total daily ration achieves this goal but also may put the young horse at risk of digestive disorders and reduced forage utilization. As such, the objective of this study was to determine if a dietary S. cerevisiae fermentation product (SCFP; TruEquine C, Diamond V) could influence nutrient utilization in young horses being introduced to grain concentrate feed and if the response was dose dependent. Thirty Quarter horse yearlings (561 ± 15.8 d of age and 371 ± 24.4 kg BW) were adapted to a 14% CP grain concentrate feed (Nutrena SafeChoice Original formulated without microbial ingredients), fed at 1% of BW as fed to meet or slightly exceed nutrient requirements for yearling horses undergoing light to moderate work and allowed ad-libitum access to Coastal bermudagrass (Cynodon dactylon) hay (67.8% NDF) and fresh water. Horses were stratified by age, BW, and sex and randomly assigned to one of 3 levels of SCFP: 0, 46, or 92 mg/kg BW divided evenly and top-dressed onto the grain concentrate meal at 12 h intervals. After 14 d of adaptation, intake and total fecal production were measured overa 4-d period. Horses were housed in individual 3.7 × 3.7 m stalls and allowed 1 h of exercise in individual 3.7 × 7.4 m runs daily during the collection period. All statistical analyses were performed in SAS 9.4 using PROC MIXED with the model containing treatment and horse included as the random variable. There was no significant treatment difference for any measure of intake (P ≥ 0.33); forage DM intake averaged 6.14 kg/d, total DM intake averaged 9.60 kg/d, and total digestible organic matter intake averaged 4.97 kg/d. There was a tendency for improved DM digestibility (P = 0.09) with supplementation (56.3 and 56.8% for 46 and 92 mg/kg BW, respectively) compared with no supplementation (54.2%). Supplementation of SCFP resulted in no significant differences (P ≥ 0.15) in organic matter (OM) or NDF digestibility. There were no differences in any indicator of nutrient digestibility between the low and high level of SCFP supplementation. An observed tendency for improved DM digestibility with SCFP supplementation suggests further research is warranted.

34 A comparison between computer vision application and a body-mounted inertial sensor system for equine gait analysis and lameness quantification

Lameness is a chief concern for horse owners; however, systems that accurately and objectively evaluate lameness are currently limited to equine veterinarians. Therefore, the objective of this study was to determine the degree of correlation in key indicators of lameness between a novel and sophisticated mobile phone computer vision (CV) application employing artificial intelligence technology versus a lameness evaluation methodology using a wireless body-mounted, inertial sensor system (ISS). The 2 methodologies were evaluated in conjunction with a study utilizing 27 Quarter horse yearlings (mean ± SEM; 561 ± 3 d). A grain-based horse feed formulated without added microbials (SafeChoice® Original, Nutrena) was fed to all horses at 1% BW/d (as-fed) twice daily in 12 h intervals for 76 d. Horses were allowed ad-libitum access to Coastal bermudagrass (Cynodon dactylon) hay and fresh water. For the 76 d period, horses were exercised on a progressive workload from moderate to heavy using mean heart rate for 30 min per d, 5d/wk using a free-stall exerciser. Horses underwent a baseline lameness assessment using ISS and CVtechnologies simultaneously on d 0 of the study, followed by 2 additional lameness assessments on d 76 (pre- exercise bout, and post- exercise bout). Data from lameness evaluations were analyzed using PROC Corr in SAS v9.4 to evaluate matching outputs (e.g., pushoff difference, head impact difference, hind pushoff difference, and hind impact difference) across both lameness detection systems. Positive correlations were noted between the ISS and CV systems when different regions were analyzed including the head pushoff difference (r = 0.87, P ≤ 0.01); head impact difference (r = 0.76, P ≤ 0.01); hind pushoff difference (r = 0.81, P ≤ 0.01); and hind impact difference (r = 0.87, P ≤ 0.01). Regardless of measurement recorded, there are strong positive relationships between the 2 systems. This suggests that the novel CV system detects and quantifies equine lameness that is highly correlated to the ISS system for several key indicators used to localize and assess degree of lameness in horses.

54 Influence of dietary Saccharomyces cerevisiae fermentation product on cartilage metabolism in young exercising horses challenged with intra-articular lipopolysaccharide

Dietary Saccharomyces cerevisiae fermentation product (SCFP) has the ability to optimize the intra-articular environment of horses, but its efficacy in young, exercising horses is not fully understood. Thirty Quarter Horse yearlings (374 ± 25 kg BW; 562 ± 16 d of age; 15 fillies, 15 geldings) were used in a 60 d study to investigate the effect of dietary SCFP (TruEquine®C, Diamond V Mills Inc.) on cartilage metabolism following an acute inflammatory insult, hypothesizing that SCFP would increase cartilage metabolism. Horses were stratified by BW, age, and sex and randomly assigned to dietary treatments (n = 10/group): 0 mg/kg BW (CON), 46 mg/kg BW SCFP (TRT1), or 92 mg/kg BW SCFP (TRT2) top-dressed to a custom-formulated concentrate void of microbials and offered at 1% BW/d (as-fed) every 12 h. On d 0, 21, 42, and 56 BW was obtained and intake adjusted accordingly. Daily grain and supplement orts were negligible across treatments. Horses were housed individually (3.6 × 7.3 m) and offered ad libitum Coastal bermudagrass hay and water. Horses were exercised on a progressive workload for 30 min/d, 5d/wk using a free-stall exerciser. On d 46, horses underwent an intra-articular lipopolysaccharide (LPS) challenge. Each horse's radial carpal joints were randomly selected to receive either 0.5 ng LPS or sterile lactated Ringer's solution (LRS) as a contralateral control.Synovial fluid obtained pre-injection (h 0) and at 6, 12, 24, and 336 h post-injection was analyzed for c-propeptide of type II collagen (CPII) and collagenase cleavage neopeptide (C2C) by commercial ELISA. Rectal temperature (RT), heart rate (HR), respiration rate (RR), and carpal circumference (CC) were recorded immediately before each arthrocentesis. Data were analyzed using PROC MIXED of SAS v9.4 and mean separation was achieved with orthogonal contrasts. The delta in BW exhibited a quadratic dose response (P ≤ 0.01) where CON horses were heavier than TRT1 and TRT2 on d 56. Diet did not influence HR, RR, or RT (P ≥ 0.25). There was an LPS × time interaction for CPII (P ≤ 0.01), CPII:C2C (P ≤ 0.01), CC (P ≤ 0.01), and a tendency in C2C (P = 0.09) for concentrations to be greater in LPS joints at 6, 12, and 24 h than LRS joints. Regardless of injection, CPII, C2C, and CPII:C2C did not return to baseline through h 336 (P ≤ 0.04). Dietary SCFP did not influence (P ≥ 0.42) biomarkers indicative of cartilage metabolism. Although, intra-articular LPS increased cartilage turnover in young exercising horses, the response was not affected by dietary SCFP.

Responses to an intra-articular lipopolysaccharide challenge following dietary supplementation of Saccharomyces cerevisiae fermentation product in young horses.

Dietary intervention may be a valuable strategy to optimize the intra-articular environment in young horses to prolong their performance career. To test the hypothesis that dietary supplementation of a Saccharomyces cerevisiae fermentation product would reduce markers of joint inflammation and increase markers of cartilage metabolism following a single inflammatory insult, Quarter Horse yearlings (mean ± SD; 9 ± 1.0 mo) were balanced by age, sex, body weight (BW), and farm of origin and randomly assigned to the following treatment groups: 1.25% BW/d (dry matter basis) custom-formulated concentrate only (CON; n = 9) or concentrate top-dressed with 21 g/d S. cerevisiae fermentation product (SCFP; n = 10) for 98 d. Horses had ad libitum access to Coastal bermudagrass hay. On day 84, one randomly selected radial carpal joint from each horse was injected with 0.5 ng lipopolysaccharide (LPS) solution. The remaining carpal joint was injected with sterile lactated Ringer’s solution as a contralateral control. Synovial fluid obtained before supplementation (day 0) and on day 84 at preinjection hour 0 and 6, 12, 24, 168, and 336 h postinjection was analyzed for prostaglandin E2 (PGE2), carboxypropeptide of type II collagen (CPII), and collagenase cleavage neopeptide (C2C) by commercial assays. Rectal temperature, heart rate, respiration rate, carpal surface temperature, and carpal circumference were recorded prior to each sample collection and for 24 h postinjection. Data were analyzed using linear models with repeated measures. From day 0 to 84, synovial C2C declined (P ≤ 0.01) and the CPII:C2C ratio increased (P ≤ 0.01) in all horses with no effect of diet. In response to intra-articular LPS, synovial PGE2 increased by hour 6 (P ≤ 0.01) and returned to baseline by hour 336; CPII increased by hour 12, remained elevated through hour 168 (P ≤ 0.01), and returned to baseline by hour 336; and C2C increased by hour 6 (P ≤ 0.01) but did not return to baseline through hour 336 (P ≤ 0.01). Post-intra-articular injection, PGE2 levels were lower in SCFP than CON horses (P = 0.01) regardless of injection type. Synovial CPII and the CPII:C2C ratio demonstrated stability during the LPS challenge in SCFP compared with CON horses (P ≤ 0.01). Clinical parameters were not influenced by diet but increased in response to repeated arthrocentesis (P ≤ 0.01). Dietary SCFP may favorably modulate intra-articular inflammation following an acute stressor and influence cartilage turnover in young horses.

Dietary supplementation of a Saccharomyces cerevisiae fermentation product attenuates exercise-induced stress markers in young horses.

Mitigation of exercise-induced stress is of key interest in determining ways to optimize performance horse health. To test the hypothesis that dietary supplementation of a Saccharomyces cerevisiae fermentation product would decrease markers of exercise-induced stress and inflammation in young horses, Quarter Horse yearlings (mean ± SD; 9 ± 1 mo) were randomly assigned to receive either no supplementation (CON; n = 8) or 21 g/d S. cerevisiae fermentation product (10.5 g/feeding twice daily; SCFP; n = 10) top-dressed on a basal diet of custom-formulated grain as well as ad libitum Coastal bermudagrass hay. After 8 wk of dietary treatments, horses underwent a 2-h submaximal exercise test (SET) on a free-stall mechanical exerciser. Serum was collected before dietary treatment supplementation (week 0), at week 8 pre-SET, and 0, 1, and 6 h post-SET and analyzed for concentrations of cortisol and serum amyloid A (SAA) by commercial enzyme-linked immunosorbent assay (ELISA) and for cytokine concentrations by commercial bead-based ELISA. Data were analyzed using linear models with repeated measures in SAS v9.4. From week 0 to 8 (pre-SET), serum cortisol decreased (P = 0.01) and SAA did not change, but neither were affected by diet. Serum concentrations of all cytokines decreased from week 0 to 8 (P ≤ 0.008), but granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and interleukin-8 (IL-8) decreased to a greater extent in CON than in SCFP horses (P ≤0.003). In response to the week 8 SET, serum cortisol increased in all horses (P < 0.0001) but returned to pre-SET levels by 1 h post-SET in horses receiving SCFP. At 6 h post-SET, cortisol concentrations in CON horses returned to pre-SET concentrations, whereas cortisol declined further in SCFP horses to below pre-SET levels (P = 0.0002) and lower than CON (P = 0.003) at that time point. SAA increased at 6 h post-SET in CON (P < 0.0001) but was unchanged through 6 h in SCFP horses. All cytokines except G-CSF increased in response to the SET (P < 0.0001) but showed differing response patterns. Concentrations of IL-1β, IL-6, and tumor necrosis factor-alpha were lesser (P ≤ 0.05), and concentrations of G-CSF and IL-18 tended to be lesser (P ≤ 0.09) in SCFP compared with CON horses throughout recovery from the SET. In summary, 8 wk of dietary supplementation with 21 g/d of SCFP may mitigate cellular stress following a single, prolonged submaximal exercise bout in young horses.

23 Forced exercise on gut microbiota and cytokines in young horses

Exercise in young horses is common but impacts on gut microbial profiles and systemic cytokines are unknown. Quarter Horse yearlings (mean ± SEM; 496 ± 12 d of age; 343 ± 28 kg BW) were used to test the hypothesis that exercise would increase beneficial gut microbial profiles and decrease serum inflammatory cytokines. Horses were balanced by age, BW, and sex and randomly assigned to non-exercise control (CON; n = 10), dry treadmill exercise (DRY; n = 10), or aquatic treadmill exercise (H2O; n = 10; water at 60% wither height). Horses were stalled 12 h/d, 5 d/wk but otherwise housed in dry lots. Horses individually received 1.25% BW/d (as-fed) commercial concentrate and ad libitum Coastal bermudagrass hay. From d0 to 112, DRY and H2O walked on treadmills 30 min/d, 5 d/wk. Workload for DRY and H2O then increased to moderate intensity on a free-stall exerciser 5 d/wk to d140. Serum and fecal samples were collected on d0, 112, and 140. Serum was analyzed for interleukin (IL)-8, IL-10, IL-12, IL-13, growth related oncogene (GRO), and tumor necrosis factor α (TNFα) concentrations via commercial bead-based ELISA. The V4 region of 16S rRNA gene was amplified from fecal community DNA using universal primers. Resulting amplicons were sequenced (2 × 250bp PE) using Illumina's NovaSeq 6000, processed using DADA2 pipeline, and analyzed using R vegan package. Significant ( P < 0.05) differences between means were determined using linear models with repeated measures, main effects of time, treatment (trt), and trt × time, and a random effect of horse(trt). Microbiome richness was greater in exercised yearlings than CON ( P < 0.02). There was a trt × time interaction for richness ( P < 0.0001); CON decreased to d112 ( P = 0.003) but increased to d140 ( P = 0.01), H2O increased to d112 ( P < 0.0001) and decreased to d140 ( P < 0.0001), while DRY increased to d140 ( P = 0.0003). Microbiome diversity increased in all horses by d112 ( P < 0.0001) and tended to decrease to d140 ( P = 0.09) but was higher than d0 on 140 ( P < 0.0001). A trend for a trt × time interaction ( P = 0.06) indicated H2O horses had greater diversity on d112 than other groups ( P ≤ 0.01). Anti-inflammatory IL-10 declined ( P < 0.0001) from d0 to 140. In contrast, pro-inflammatory GRO, TNFα, and IL-8 increased from d0 to 112 ( P ≤ 0.008). From d112, GRO plateaued to d140 to remain greater than d0 at 140 ( P = 0.01), TNFα did not differ between d0 and 140, and IL-8 decreased to d140 ( P = 0.02). Cytokines did not differ by trt. Exercise improved microbial richness but did not impact microbial diversity or systemic cytokines. Future research should explore the effects of more intense exercise, diet, and environment on gut health and systemic cytokines in young horses.

114 Effects of aquatic conditioning in young horses. II. Bone metabolism

Abstract While beneficial in rehabilitation, aquatic exercise effects on bone metabolism in young, healthy horses has not been investigated. Therefore, thirty Quarter Horse yearlings were stratified by age, body weight (BW), and sex and randomly assigned to one of three treatments during a 140-d trial to evaluate aquatic vs. dry exercise on bone metabolism in young horses transitioning to an advanced workload. Treatment groups included non-exercise control (CON; n = 10), dry treadmill exercise (DRY; n = 10), or aquatic treadmill exercise (H2O; n = 10; water: 60% wither height, WH). Animals were housed in individual stalls (3.6 m×3.6 m) from 0600 to 1800, allowed turnout (74 m×70 m) from 1800 to 0600, and fed to meet or exceed requirements. During Phase I, DRY and H2O walked on treadmills 30 min/d, 5 d/wk from d 0–112. Phase II transitioned to an advanced workload 5 d/wk for 28 d (Table 1). Every 14 d, WH, hip height (HH), and BW were recorded. Every 28 d following exercise, serum samples were collected for osteocalcin (OC) and C-telopeptide crosslaps of type I collagen (CTX-1) analysis. Left third metacarpal radiographs on d 0, 112, and 140 were analyzed for radiographic bone aluminum equivalence (RBAE). Data were analyzed using PROC MIXED of SAS. Baseline treatment differences in biomarkers were accounted for using a covariate. There were treatment ′ day interactions (P &amp;lt; 0.01) where OC and CTX-1 remained consistent in both exercise groups while inconsistently increasing in CON. There were no treatment differences (P &amp;gt; 0.30) in RBAE, BW, or HH, but all increased over time (P &amp;lt; 0.01). There was a tendency toward a treatment × day interaction for WH (P = 0.07), characterized by a difference in response by CON during the first 28 d. This study indicates that early forced exercise supports consistent bone metabolism necessary for uniform growth and bone development, while lack of forced exercise results in incongruent bone turnover.

113 Effects of aquatic conditioning in young horses. I. Markers of inflammation and cartilage metabolism

Abstract Aquatic treadmills improve range of motion and increase muscular strength in mature horses; however, effects of buoyancy on inflammation and cartilage metabolism in young horses are not well investigated. Therefore, thirty Quarter Horse yearlings of similar breeding were stratified by age, BW, and sex and randomly assigned to one of three treatment groups during a 140-d trial to evaluate the influence of aquatic vs. dry exercise on joint inflammation and cartilage metabolism in young horses transitioning to an advanced workload. Treatment groups included non-exercise control (CON; n = 10), dry treadmill exercise (DRY; n = 10), or aquatic treadmill exercise (H2O; n = 10; water at 60% wither height). Animals were housed in individual stalls and allowed turnout for a minimum of 10 h/d. During Phase I, DRY and H2O walked on treadmills 30 min/d, 5 d/wk from d 0 to d 112. Phase II represented transition to an advanced workload 5d/wk for 28 d (Table 1). Every 28 d following exercise, synovial fluid samples were collected and analyzed for prostaglandin E2 (PGE2), collagenase cleavage neopeptide (C2C), collagenase of type I and type II collagen (C1,2C), and carboxypeptide of type II collagen (CPII) using commercial ELISA kits. All data were analyzed using PROC MIXED of SAS, including random effect of horse within treatment, and repeated effect of day. Baseline treatment differences were accounted for using a covariate structure. There were no treatment ′ day interactions of synovial inflammation or markers of cartilage metabolism; however, there was an effect of day for each selected marker (P &amp;lt; 0.03). Changes in biomarkers of cartilage turnover in horses exercised at the walk, whether dry or aquatic, could not be distinguished from horses with access to turnout alone. This indicates that there are no negative effects of buoyancy on cartilage metabolism in yearlings transitioned from aquatic exercise to 28-d advanced workload.

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

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

Submaximal exercise training improves mitochondrial efficiency in the gluteus medius but not in the triceps brachii of young equine athletes

We tested the hypothesis that, similar to humans and rodents, exercise training would enhance mitochondrial (Mt) biogenesis and function in skeletal muscle of young horses. Twenty-four Quarter Horse yearlings were randomly assigned to either submaximal exercise training or no forced exercise (untrained). Biopsies were collected from the gluteus medius and triceps brachii before and after 9 wk of treatment. Citrate synthase activity was lower (P < 0.0001) and cytochrome c oxidase activity per Mt unit was higher (P < 0.0001) in gluteus compared to triceps, but neither changed over the trial period. From wk 0 to 9, intrinsic Mt respiration (PCI, PCI+II; P = 0.008) and electron transport capacity (ECI+II; P = 0.01) increased, and LEAK-related flux control factor (FCFL; P = 0.02) decreased in both muscles. After 9 wk of training, gluteus muscle exhibited higher (P < 0.05) intrinsic PCI, PCI+II, ECI+II, and FCFCI and FCFCI+II, and lower FCFL (P = 0.0002). Mitochondrial content did not change from wk 0 to 9, and also not in response to submaximal exercise training. Improvements in Mt function were most directly related to ongoing growth of horses independent of muscle group, and training further enhanced Mt function in the gluteus medius.

Rapid Communication: Dietary selenium improves skeletal muscle mitochondrial biogenesis in young equine athletes1

Exercise is known to promote mitochondrial biogenesis in skeletal muscle as well as enhance mitochondrial function and efficiency in human and rodent models. These adaptations help to decrease exercise-associated production of reactive oxygen species, which can negatively affect health and performance if antioxidant mechanisms are overwhelmed. Little is known about the adaptations of mitochondria in response to exercise training in the growing horse or if supplementation with a dietary antioxidant can improve mitochondrial function. To evaluate the separate and combined effects of selenium (Se) supplementation, training, and an acute strenuous exercise bout on mitochondrial adaptations in young horses, 30 American Quarter Horse yearlings were randomly assigned to an exercise training group or a no-training group and, within each group, received either 0.1 or 0.3 mg Se/kg DM for 14 wk. The study was split into 2 phases (wk 0 to 8 and wk 9 to 14), with half of the trained horses switched to the opposite dietary treatment in Phase 2. At the end of each phase, all horses underwent a 120-min submaximal exercise test (SET; SET 1 and SET 2). Biopsies of the middle gluteal muscle were collected before and after each phase of the study and in response to each SET and analyzed for markers of mitochondrial number and function. At rest, horses receiving 0.3 mg Se/kg DM had higher citrate synthase activity (P = 0.021) than horses receiving 0.1 mg Se/kg DM, indicating higher mitochondrial content. In contrast, cytochrome c oxidase (CCO) activity was not affected by dietary Se overall, but horses that were dropped from 0.3 mg Se/kg DM to 0.1 mg Se/kg DM during Phase 2 showed a decrease (P = 0.034) in integrated CCO activity from wk 9 to 14, suggesting impaired mitochondrial function. Mitochondrial enzyme activities were unaffected by an acute, strenuous exercise bout (SET 1 and SET 2). Our relatively low-intensity exercise training protocol did not appear to induce functional mitochondrial adaptations. However, elevated dietary Se may impart beneficial effects on mitochondrial biogenesis during growth and training. A more strenuous exercise training protocol should be investigated to determine the potential benefits of elevated dietary Se for elite equine athletes.

Submaximal exercise training, more than dietary selenium supplementation, improves antioxidant status and ameliorates exercise-induced oxidative damage to skeletal muscle in young equine athletes.

Exercise is associated with increased production of reactive oxygen species (ROS) as metabolism is upregulated to fuel muscle activity. If antioxidant systems become overwhelmed, ROS can negatively affect health and performance. Adaptation to exercise through regular training has been shown to improve defense against oxidative insult. Given selenium's role as an antioxidant, we hypothesized that increased Se intake would further enhance skeletal muscle adaptations to training. Quarter Horse yearlings (18 ± 0.2 mo; 402 ± 10 kg) were randomly assigned to receive either 0.1 or 0.3 mg Se/kg DM and placed in either an untrained or a trained (30 min walk-trot-canter, 4 d/wk) group for 14 wk. Phase 1 (wk 1 to 8) consisted of 4 treatments: trained and fed 0.1 mg Se/kg DM through wk 14 (CON-TR; n = 10), trained and fed 0.3 mg Se/kg DM through wk 14 (HIGH-TR; n = 10), untrained and fed 0.1 mg Se/kg DM through wk 14 (CON-UN; n = 5), or untrained and fed 0.3 mg Se/kg DM through wk 14 (HIGH-UN; n = 5). During Phase 2 (wk 9 to 14), dietary Se level in half of the trained horses was reversed, resulting in 6 treatments: CON-TR (n = 5), trained and fed 0.1 mg/kg Se in Phase 1 and then switched to 0.3 mg/kg Se for Phase 2 (ADD-TR; n = 5), trained and fed 0.3 mg/kg Se in Phase 1 and then switched to 0.1 mg/kg Se for Phase 2 (DROP-TR; n = 5), HIGH-TR (n = 5), CON-UN (n = 5), or HIGH-UN (n = 5). All horses underwent a 120-min submaximal exercise test (SET) at the end of Phase 1 (SET 1) and 2 (SET 2). Blood samples and biopsies from the middle gluteal muscle were collected before and after each phase of the study and in response to each SET and analyzed for markers of oxidative damage and antioxidant enzyme activity. In both phases, serum Se was higher (P < 0.0001) when horses received a diet with 0.3 than 0.1 mg Se/kg DM. Throughout the 14-wk study, resting activities of muscle glutathione peroxidase (GPx; P = 0.004) and superoxide dismutase (SOD; P = 0.06) were greater in trained horses than in untrained horses. In response to SET 1, serum creatine kinase (CK) activity was lower in trained horses than in untrained horses (P < 0.0001), indicating less muscle damage, but plasma lipid hydroperoxides (LPO) and muscle GPx and SOD activities were unaffected by training or Se. In response to SET 2, trained horses had greater muscle SOD activity (P = 0.0002) and lower serum CK activity (P = 0.003) and showed a trend for lower plasma LPO (P = 0.09) and muscle malondialdehyde (P = 0.09) than untrained horses. Muscle GPx activity did not change in response to SET 2 and was unaffected by training or Se. Results indicate that exercise training lessens muscle damage and improves antioxidant defense following an acute bout of prolonged exercise and was not further enhanced by feeding Se above the NRC requirement.

Rapid Communication: Dietary selenium improves skeletal muscle mitochondrial biogenesis in young equine athletes.

Exercise is known to promote mitochondrial biogenesis in skeletal muscle as well as enhance mitochondrial function and efficiency in human and rodent models. These adaptations help to decrease exercise-associated production of reactive oxygen species, which can negatively affect health and performance if antioxidant mechanisms are overwhelmed. Little is known about the adaptations of mitochondria in response to exercise training in the growing horse or if supplementation with a dietary antioxidant can improve mitochondrial function. To evaluate the separate and combined effects of selenium (Se) supplementation, training, and an acute strenuous exercise bout on mitochondrial adaptations in young horses, 30 American Quarter Horse yearlings were randomly assigned to an exercise training group or a no-training group and, within each group, received either 0.1 or 0.3 mg Se/kg DM for 14 wk. The study was split into 2 phases (wk 0 to 8 and wk 9 to 14), with half of the trained horses switched to the opposite dietary treatment in Phase 2. At the end of each phase, all horses underwent a 120-min submaximal exercise test (SET; SET 1 and SET 2). Biopsies of the middle gluteal muscle were collected before and after each phase of the study and in response to each SET and analyzed for markers of mitochondrial number and function. At rest, horses receiving 0.3 mg Se/kg DM had higher citrate synthase activity ( = 0.021) than horses receiving 0.1 mg Se/kg DM, indicating higher mitochondrial content. In contrast, cytochrome oxidase (CCO) activity was not affected by dietary Se overall, but horses that were dropped from 0.3 mg Se/kg DM to 0.1 mg Se/kg DM during Phase 2 showed a decrease ( = 0.034) in integrated CCO activity from wk 9 to 14, suggesting impaired mitochondrial function. Mitochondrial enzyme activities were unaffected by an acute, strenuous exercise bout (SET 1 and SET 2). Our relatively low-intensity exercise training protocol did not appear to induce functional mitochondrial adaptations. However, elevated dietary Se may impart beneficial effects on mitochondrial biogenesis during growth and training. A more strenuous exercise training protocol should be investigated to determine the potential benefits of elevated dietary Se for elite equine athletes.

Effects of Dietary Selenium and Training on Mitochondrial Function in Young Horses

IntroductionExercise training results in improved mitochondrial efficiency but it is unclear if dietary selenium level can further modify mitochondrial function.MethodsAmerican Quarter Horse yearlings (n = 20; mean ± SE 18 ± 0.2 months) balanced with regard to age and sex were randomly assigned to four dietary Se and exercise training treatments for 14 weeks: 0.1 mg Se/kg DM with (n = 5) or without (n = 5) training or 0.3 mg Se/kg DM with (n = 5) or without (n = 5) training. Trained horses were exercised 4 d/wk for 30 min/d. At week 14, all horses underwent a 2 hr, 27.5 km submaximal exercise test (SET). Permeabilized muscle fibers obtained from the gluteus medius of horses at rest were analysed for mitochondrial function via high resolution respirometry. Mitochondrial oxidative phosphorylation capacity with Complex I substrates was determined using the respiratory control ratio (RCR) of state 3 (with ADP) and state 2 (without ADP; leak respiration). Electron transport system capacity (ETSC) was determined by uncoupling phosphorylation and respiration using carbonyl cyanide p‐trifluoromethoxyphenylhydrazone. Cytochrome c oxidase (CCO) and citrate synthase (CS) activities were assessed in resting muscle samples obtained at week 0 and 14, and in response to the SET. Data were analysed using a mixed model ANOVA.ResultsLeak, state 3, ETSC, CS and CCO were higher (P&lt;0.05) at wk 14 compared to wk 0 in all horses, but were not affected by training or Se level. Additionally, CS and CCO were unaffected by the SET.ConclusionsMitochondrial number (CS activity) and efficiency were unaffected by training and dietary Se level, but did increase over time, perhaps due to growth of horses. A more strenuous training protocol may be needed to induce mitochondrial adaptations.Ethical Animal ResearchThis project was approved by the Institute of Food and Agricultural Sciences Animal Care Committee at the University of Florida (approval number 016‐12ANS). Sources of funding: Partial financial support of this project was provided by Alltech, Nicholasville, KY, USA. Competing interests: none.

Effects of Dietary Selenium and Training on Oxidative Stress in Young Horses

IntroductionExercise training can enhance antioxidant status, but it is unknown whether selenium supplementation can further improve antioxidant defenses.MethodsAmerican Quarter Horse yearlings (n = 20; mean ± SE 18 ± 0.2 months) balanced with regard to age and sex were randomly assigned to four dietary Se and training treatments for 14 weeks: 0.1 mg Se/kg DM with (n = 5) or without (n = 5) training or 0.3 mg Se/kg DM with (n = 5) or without (n = 5) training. Trained horses were exercised 4 d/wk for 30 min/d. At wk 14, all horses underwent a 2‐hr, 27.5 km submaximal exercise test (SET). Blood samples and biopsies of the gluteus medius were obtained before and after training and in response to the SET. Serum Se, glutathione peroxidase (GPx) activity in whole blood and muscle, and plasma lipid hydroperoxides (LPO) were evaluated. Data were analysed using a mixed model ANOVA with repeated measures.ResultsSerum Se was higher (P = 0.02) at wk 14 in trained horses receiving 0.3 than 0.1 mg Se/kg, but was unaffected by diet in untrained horses. Muscle GPx activity was unchanged after 14‐weeks of Se and training, but in response to the SET, muscle GPx was higher (P = 0.05) in trained than untrained horses. An effect of training*time (P = 0.008) was noted for whole blood GPx, reflecting greater (P = 0.01) activity in trained than untrained horses immediately after the SET. Plasma LPO was not influenced by dietary Se, but increased (P&lt;0.0001) in untrained horses immediately after the SET while remaining unchanged in trained horses.ConclusionsTraining appears to mitigate oxidative damage in response to prolonged exercise in young horses. Although trained horses responded to a higher level of dietary Se, it showed no clear advantage for reducing oxidative stress.Ethical Animal ResearchThis project was approved by the Institute of Food and Agricultural Sciences Animal Care Committee at the University of Florida (approval number 016‐12ANS). Sources of funding: Partial financial support of this project was provided by Alltech, Nicholasville, KY, USA. Competing interests: none.

Intradermal injections of equine allogeneic umbilical cord-derived mesenchymal stem cells are well tolerated and do not elicit immediate or delayed hypersensitivity reactions

BACKGROUND AIMS. The use of allogeneic mesenchymal stem cells (MSC) to treat acute equine lesions would greatly expand equine cellular therapy options; however, the safety and antigenicity of these cells have not been well-studied. We hypothesized that equine allogeneic umbilical cord tissue (UCT)-derived MSC would not elicit acute graft rejection or a delayed-type hypersensitivity response when injected intradermally. METHODS. Six Quarterhorse yearlings received 12 intradermal injections (autologous MSC, allogeneic MSC, positive control and negative control, in triplicate) followed by the same series of 12 injections, 3-4 weeks later, at another site. Wheals were measured and palpated at 0.25, 4, 24, 48, 72 h and 7 days post-injection. Biopsies were obtained at 48 and 72 h and 7 days post-injection. Mixed leukocyte reactions were performed 1 week prior to the first injections and 3 weeks after the second injections. RESULTS. There were no adverse local or systemic responses to two intradermal injections of allogeneic MSC. MSC injection resulted in minor wheal formation, characterized by mild dermatitis, dermal edema and endothelial hyperplasia, that fully resolved by 48-72 h. No differences were noted between allogeneic and autologous MSC. The second injection of MSC did not elicit more significant physical or histomorphologic alterations compared with the first MSC injection. Neither allogeneic nor autologous UCT-derived MSC stimulated or suppressed baseline T-cell proliferation in vitro prior to or after two MSC administrations. CONCLUSIONS. Equine allogeneic UCT MSC may be safely administered intradermally on multiple occasions without eliciting a measurable cellular immune response.

Supplemental leucine's influence on plasma glucose, insulin, and amino acid responses in Quarter Horse yearlings

Supplemental leucine's influence on plasma glucose, insulin, and amino acid responses in Quarter Horse yearlings

Effect of dietary omega-3 fatty acid source on plasma and red blood cell membrane composition and immune function in yearling horses1

To determine the effect of different sources of dietary n-3 fatty acids (FA) on plasma and red blood cell (RBC) FA composition and immune response, 18 Quarter Horse yearlings were randomly and equally assigned to 1 of 3 treatments: encapsulated fish oil (n = 6), milled flaxseed (n = 6), or no supplementation (control, n = 6). Fish oil contained 15 g of eicosapentaenoic acid (C20:5n-3) and 12.5 g of docosahexaenoic acid (C22:6n-3), and flaxseed contained 61 g of alpha-linolenic acid (C18:3n-3) per 100 g of FA. Horses had free access to bahiagrass pasture during the active growing season and were individually fed a grain mix concentrate at 1.5% BW/d. Fish oil and flaxseed were mixed into the concentrate in amounts to provide 6 g of total n-3/100 kg of BW. Horses were fed their respective treatments for 70 d. Blood samples were obtained to determine plasma and RBC FA composition and for isolation of peripheral blood mononuclear cells. Peripheral blood mononuclear cells were stimulated with concanavalin A and phytohemagglutinin (PHA) to determine lymphocyte proliferation and were challenged with lipopolysaccharide to determine PGE(2) production. In vivo inflammatory response was assessed on d 70 by measuring skin thickness and area of swelling in response to intradermal injection of PHA. Treatment did not affect BW gain, which averaged 0.6 +/- 0.03 kg/d. Horses fed fish oil had greater (P < 0.05) proportions of eicosapentaenoic acid, docosahexaenoic acid, and sum of n-3 in plasma and RBC compared with those in the flaxseed and control treatments. In addition, plasma arachidonic acid was greater (P < 0.05) and plasma linoleic and alpha-linolenic acids were less (P < 0.05) in the fish oil treatment compared with the flaxseed and control treatments. Dietary treatment did not affect lymphocyte proliferation or PGE(2) production. Across treatments, the peak increase in skin thickness was observed 4 to 8 h after PHA injection. At 4 h postinjection, horses fed fish oil and those fed flaxseed had a greater increase in skin thickness than those in the control treatment (P < 0.05) and horses fed fish oil had a larger area of swelling than those in the control treatment (P < 0.05). Skin thickness remained greater (P < 0.05) in horses fed flaxseed than in control horses 6 h after injection. Although fed to supply a similar amount of n-3 FA, fish oil had a greater impact on plasma and RBC n-3 FA content than did flaxseed. However, supplementing horses with both fish oil and flaxseed resulted in a more pronounced early inflammatory response to PHA injection as compared with nonsupplemented horses.

Effects of Mineral Source on Growth and Balance in Yearling Horses1

Sixteen Quarter-Horse yearlings were used in a split-plot designed experiment to evaluate the response of growth and mineral balance to trace mineral source. Horses were blocked by sex and BW and randomly assigned to either an inorganic (ING) or organic (ORG) treatment. Diets were formulated to contain supplemental levels of Zn, Cu, Mn, and Co at four times NRC (1989) recommendations for yearlings in rapid growth. Diets were fed at 2% of estimated final BW and consisted of a pelleted concentrate, Marshall ryegrass hay, and alfalfa cubes fed in a 50:30:20 ratio, respectively. Three 72-h total fecal and urine collections were conducted on d 7, 30, and 60. Blood was taken on d 1 of each total collection to measure serum mineral (Zn and Cu) concentrations. Although mineral digestibility and balance data were not consistent across periods, significant increases in Cu (Period II) and Co (Periods II and III) digestibility and balance were observed for the ORG treatment. These increases indicate that ORG trace minerals may be more available for absorption as compared with IN sources, although excessive supplementation of trace minerals in the diet may obscure differences because of trace mineral source. Thus, the feeding of ORG trace minerals may improve trace mineral absorption, subsequently improving balance and digestibility.