Quarter Horses Research Articles

Increased α-tocopherol metabolism in horses with equine neuroaxonal dystrophy.

BackgroundEquine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with a vitamin E deficiency within the first year of life. Vitamin E consists of 8 isoforms metabolized by the CYP4F2 enzyme. No antemortem diagnostic test currently exists for eNAD/EDM.Hypothesis/ObjectivesBased on the association of α‐tocopherol deficiency with the development of eNAD/EDM, we hypothesized that the rate of α‐tocopherol, but not γ‐tocopherol or tocotrienol metabolism, would be increased in eNAD/EDM‐affected horses.AnimalsVitamin E metabolism: Proof of concept (POC) study; eNAD/EDM‐affected (n = 5) and control (n = 6) horses. Validation study: eNAD/EDM‐affected Quarter Horses (QHs; n = 6), cervical vertebral compressive myelopathy affected (n = 6) horses and control (n = 29) horses. CYP4F2 expression and copy number: eNAD/EDM‐affected (n = 12) and age‐ and sex‐matched control (n = 11‐12) horses.MethodsThe rates of α‐tocopherol/tocotrienol and γ‐tocopherol/tocotrienol metabolism were assessed in equine serum (POC and validation) and urine (POC only) using liquid chromatography tandem mass spectrometry (LC‐MS/MS). Quantitative reverse‐transcriptase PCR (qRT‐PCR) and droplet digital (dd)‐PCR were used to assay expression and genomic copy number of a CYP4F2 equine ortholog.ResultsMetabolic rate of α‐tocopherol was increased in eNAD/EDM horses (POC,P < .0001; validation, P = .03), with no difference in the metabolic rate of γ‐tocopherol. Horses with eNAD/EDM had increased expression of the CYP4F2 equine orthologue (P = .02) but no differences in copy number.Conclusions and Clinical ImportanceIncreased α‐tocopherol metabolism in eNAD/EDM‐affected QHs provides novel insight into alterations in vitamin E processing in eNAD/EDM and highlights the need for high‐dose supplementation to prevent the clinical phenotype in genetically susceptible horses.

Effect of Donor Diet on In Vitro Digestibility of Forages by Fecal Inoculate

Previous research indicates equine fecal inoculates produce comparable results to cecal fluid when used for in vitro procedures to analyze dry matter digestibility (DMD). Equine hindgut microbial communities represented in fecal samples have been shown to be affected by diet. The study's objective was to determine the effect of the donor diet on in vitro DMD when fecal donors were fed high starch, or high fiber diets. Six Quarter Horses were used in a crossover design to compare the effects of a grain versus forage diet on in vitro digestion of forages ranging from: CP 7.7 to 16.4 %DM, NDF 53 to 72 %DM. Feces from each horse were obtained on day 22 of each period and used to inoculate in vitro fermentation vessels in order to evaluate the effect of donor diet on the DMD, neutral detergent fiber digestibility (NDFD), and acid detergent fiber digestibility (ADFD) of four forages. Data were analyzed using the MIXED procedure of SAS to evaluate digestibility differences in the diet by forage composition interaction. Fecal samples from horses on the grain diet promoted higher NDFD of forages with high NDF and low CP when CP and NDF were used as covariates (P=.04 and .03). There was a horse effect on DMD and NDFD (P ≤ .05). Findings suggest diet may influence the hindgut microbiome's ability to digest neutral detergent fiber and should be considered when selecting equine fecal samples for in vitro digestion procedures.

Slab fractures of the third carpal bone involving both radial and intermediate facets and outcomes after surgical repair in racing Quarter Horses.

Characteristics and outcomes after surgical repair of third carpal bone (C3) slab fractures involving both radial and intermediate facets in racing Quarter Horses are unknown. To describe the pre- and intraoperative characteristics of C3 slab fractures of both radial and intermediate facets in Quarter Horses and to report on the long-term outcomes after internal fixation. Retrospective case series. Case records were collected from racehorses with C3 slab fractures between 2008 and 2020. Inclusion criteria required arthroscopic-guided repair of C3 slab fractures involving both radial and intermediate facets in Quarter Horses. Routine C3 slab fractures (single facet), fractures in other breeds or those repaired with other techniques were excluded. Outcomes were obtained by standardised questionnaire. Data were presented as mean±SD or as proportions with 95% confidence interval (CI). Of 22 Quarter Horses with C3 slab fractures involving both radial and intermediate facets, 91% (CI 79%-100%; n=20) were collapsing and 91% (CI 79%-100%; n=20) had avulsion of the medial palmar intercarpal ligament. Articular cartilage erosion and osteochondral fracture of the radial carpal bone was observed in 91% (CI 79%-100%; n=20) and 41% (CI 20%-62%; n=9) cases respectively. At 5.5±3.9years after surgery, 86% (CI 72%-100%; n=19) were alive and used for breeding or retirement. Of 18 horses with follow-up >1year, 39% (CI 16%-61%; n=7) resumed some athletic activity. Cases were referred specifically for surgical repair and horses with fractures considered too severe for surgical intervention or euthanasia at owner request were not included. Questionnaire responses are susceptible to recall bias. Horses with C3 slab fractures of both radial and intermediate facets that are repaired have a favourable prognosis for retirement, breeding and potentially low-level athletic activity.

Postmortem diagnoses of spinal ataxia in 316 horses in California.

To determine period prevalences of postmortem diagnoses for spinal cord or vertebral column lesions as underlying causes of ataxia (spinal ataxia) in horses. 2,861 client-owned horses (316 with ataxia [ataxic group] and 2,545 without ataxia [control group]). The medical records database of the University of California-Davis Veterinary Medical Teaching Hospital was searched to identify horses necropsied between January 1, 2005, and December 31, 2017. Results were compared between the ataxic and control groups and between various groups of horses in the ataxic group. Period prevalences were determined for the most common causes of ataxia. 2,861 horses underwent full necropsy, and the period prevalences for the most common definitive diagnoses for ataxia were 2.7% (77/2,861) for cervical vertebral compressive myelopathy (CVCM), 1.3% (38/2,861) for equine neuroaxonal dystrophy or equine degenerative myeloencephalopathy (eNAD-EDM), and 0.9% (25/2,861) for trauma; the period prevalence of ataxia of unknown origin was 2.0% (56/2,861). Horses in the ataxic group (vs the control group) were more likely to have been warmblood horses (OR, 2.70) and less likely to have been Arabian horses (OR, 0.53). In the ataxic group, horses < 5 (vs ≥ 5) years of age had greater odds of CVCM (OR, 2.82) or eNAD-EDM (OR, 6.17) versus trauma or ataxia of unknown origin. Horses in the ataxic group with CVCM were more likely Thoroughbreds (OR, 2.54), whereas horses with eNAD-EDM were more likely American Quarter Horses (OR, 2.95) and less likely Thoroughbreds (OR, 0.11). Results indicated that breed distributions differed for horses with CVCM versus eNAD-EDM; therefore, breed should be considered in the clinical evaluation of spinal ataxia in horses.

Dietary omega-3 fatty acid supplementation does not impair vitamin E status or promote lipid peroxidation in growing horses.

Omega-3 (n-3; ω-3) fatty acids (FA) are often included in the diet for their potential health benefits. However, because oxidative potential is increased with the degree of unsaturation in vitro, polyunsaturated FA such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) may be at increased risk of lipid peroxidation. We aimed to determine the effects of dietary n-3 FA supplementation on antioxidant status and lipid peroxidation in yearling horses. Quarter Horses (mean ± SEM; 14.6 ± 0.2 mo) were randomly assigned to receive no n-3 FA supplementation (CON; n = 6) or 60 mg n-3/kg body weight from milled flaxseed (FLAX; n = 6) or encapsulated fish oil (FISH; n = 6). All horses received a basal diet of mixed grain concentrate fed individually at 1.5% body weight (dry matter basis) and ad libitum bahiagrass pasture forage. Blood samples were obtained before and after 70 d of supplementation to evaluate vitamin E, selenium, lipids, antioxidant status, and oxidative stress. Data were analyzed using a mixed model ANOVA with repeated measures. Supplementation with n-3 FA did not reduce serum vitamin E or Se and, in fact, elevated (P ≤ 0.0003) vitamin E status in FISH horses. At day 70, serum triglycerides were lower in FISH and FLAX horses than CON horses (P ≤ 0.02) and F2-isoprostanes were lower in FISH than CON horses (P = 0.0002). Dietary n-3 FA had no effect on cholesterol, reduced and oxidized glutathione, glutathione peroxidase, and thiobarbituric acid-reactive substances. In growing horses fed to meet their vitamin E requirements, supplementation with 60 mg n-3/kg body weight did not negatively affect vitamin E status or promote lipid peroxidation. Elevated vitamin E status in horses fed FISH, coupled with lower serum F2-isoprostanes, further suggest that the longer-chain, highly unsaturated n-3 FA, EPA and DHA, may actually attenuate lipid peroxidation.

144 Lack of correspondence between commercial genetic test variants for type 2 polysaccharide storage myopathy and a histopathologic diagnosis in Quarter Horses

A diagnosis of type 2 polysaccharide storage myopathy (PSSM2) in Quarter Horse-related breeds (QH) is currently based on the absence of the glycogen synthase 1 ( GYS1) mutation causing type 1 PSSM (PSSM1) and presence of aggregates polysaccharide in myofibers. Recently, genetic tests for PSSM2 have been offered by a commercial company based on variants in the genes myotilin (termed P2), filamin C (P3) and myozenin 3 (P4). The company claims these variants are associated with PSSM2 as phenotyped by clinical signs without regard to muscle histopathology. The purpose of this study was to compare genotype and allele frequencies of commercial test variants (P variants) in MYOT (P2; rs1138656462), FLNC (P3a; rs1139799323 and P3b; rs1142918816) and MYOZ3 (P4; rs1142544043) between Quarter Horses diagnosed with PSSM2 by muscle histopathology and healthy Quarter Horse controls with normal muscle histology (periodic acid Schiff's stain). DNA was isolated from hair bulb, buffy coat, whole blood or muscle tissue samples. Primers were designed and genotyping performed using pyrosequencing. Genotypes at each locus (homozygous reference vs. heterozygous, homozygous alternate) were compared between PSSM2 (n = 54, 7.6 ± 4.2 y) and control horses (n = 121, 9.8 ± 5.7 y) using a Fisher's exact test ( P ≤ 0.05). There was no significant association between any P locus and a histopathological diagnosis of PSSM2. Minor allele frequencies (cases/controls) were: 20%/27% (P2), 13%/10% (P3a), 11%/9% (P3b)/and 11%/10% (P4). The P2 variant was present in 48% of control and 33% of PSSM2 Quarter Horses. In conclusion, the P2, P3a, P3b and P4 commercial test variants appear to be relatively common variants in Quarter Horses not associated with a diagnosis of PSSM2 established by muscle biopsy.

145 Comparison of skeletal muscle citrate synthase activity across equine breeds

Skeletal muscle citrate synthase (CS) activity serves as a marker for muscle oxidative capacity. CS activity was compared among Quarter Horses (QH), Arabians (AR), Thoroughbreds (TB), Standardbreds and Warmbloods at various ages and stages of training. Gluteus medius muscle biopsies were obtained at ages 6–23 m (n = 10), 2 y (n = 37), 3 y (n = 44) and >3 y (n = 55). Disciplines included reining (QH n = 17), ≥ 50-mile endurance (AR n = 20), racing (TB n = 47, STD n = 14) and dressage (WB n = 15). CS activity was measured fluorometrically. An unpaired t -test or one-way ANOVA compared age groups within breeds and age groups across breeds. A 2-way ANOVA compared breed and age effects across 2 and 3 y-old QH and TB, 3 and 4–7 y-old WB (insufficient numbers of 2 y-old AR and STD). CS was significantly lower in 2 y-old QH than either 2 y-old TB (43% higher, P < 0.0001) or 3 y-old WB (12%, P = 0.018). CS did not differ between 3 y-old QH and 3 y-old TB and was higher in 3 y-old TB than 4–7 y-old WB ( P = 0.046). Within breeds, CS was lower in <2 versus 3 y in QH (44% lower, P = 0.001), 3–6 y in TB (35%, P = 0.02) and 6–19 y in AR (30%, P = 0.051) but not versus ≥5 y old WB (9%, P = 0.39). In all horses in full training, CS activity was not significantly different across breeds ( P = 0.099), with mean CS in QH being 20–24% lower versus TB and STD, 17% lower than AR and 4% lower than in WB, and wide individual variation apparent. In conclusion, in horses ≤2 y of age, differences exist in aerobic capacity between breeds. However, effects of age, training and individual variation obscure breed differences in muscle CS activity in horses >3 y in training.

138 Expression profiling of the ventral colon, pelvic flexure, and dorsal colon epithelium by RNA-seq

The equine large intestine is essential for nutrient absorption and as a location for microbial digestion. The pelvic flexure is an important anatomical landmark that divides distinct microbial communities in the equine hindgut. Previous work from our group showed that several taxonomic groups were restricted to the cecum and ventral colon. Since no physical barrier separates these compartments from the more distal areas, we became curious about alternative mechanisms that support the segregation of different microbial groups. Physiological differences in each compartment likely result in different requirements from the microbiota at each location. This study evaluated gene expression in the mucosal and submucosal layers of the right and left ventral colon (VC), pelvic flexure (PF), and right and left dorsal colon (DC). Samples were collected from 3 4-year-old American Quarter Horses, and total RNA was isolated. Single-end libraries were prepared using the NEBNextⓇ Ultra II Directional RNA library kit and sequenced on an IlluminaⓇ NextSeq 550 platform. The sequencing generated an average of 22.5 million reads per sample (range 14.5 to 30.9). Gene expression analysis was performed using the Galaxy platform by aligning quality trimmed sequence reads to EquCab3 using the HISAT2, assembling transcripts with StringTie, and quantifying transcript abundance with Salmon. DESeq2 was used to assess differential expression between the intestinal compartments. Expression (present in 2 of 3 biological replicates) was detected in at least one intestinal compartment for 21,987 transcripts and ranging from 14,766 to 15,134 transcripts per sample. A total of 13,944 transcripts were detected in all 5 areas, and 739 transcripts demonstrated a tissue-restricted expression pattern (detected above threshold in only on anatomical location). Gene ontology was used to identify the functional roles of transcripts expressed in all or only one hindgut compartment. Transcripts expressed in all tissues were associated with internal cell metabolic processes, which are common despite cell type. In contrast, transcripts with tissue-restricted expression patterns were associated with cellular differentiation and secretion processes. Matrix metalloproteinases, transcription factors, and integrin proteins were identified as differentially expressed (P < 0.05) in the PF compared with the surrounding VC and DC compartments. Continued investigation of these transcripts in the equine hindgut will enhance our understanding of digestive physiology and support future studies of interactions between host and microbiota.

40 Dietary Saccharomyces cerevisiae fermentate affects mucosal immunity in young stress-challenged horses in training

Competition horses are under pressure from a wide range of stressors putting them at higher risk of infectious disease. Ingestion of Saccharomyces cerevisiae fermentate (SCF) products have been shown to improve immunocompetence in humans and other species, and we hypothesized it would provide similar benefits for horses. Yeast fermentate contains cellular components that can bind to pathogens and stimulate host immune cells, as well as bioactive fermentation byproducts (e.g., short chain fatty acids, vitamins, antioxidants) that can augment gut and other mucosal defenses. This study aimed to evaluate the effect of SCF on mucosal immunity in young horses in training. Two-year-old Quarter Horses (mean ± SEM; initial age 22 ± 0.3 mo and BW 439 ± 3 kg) were paired by age and sex and randomly assigned to receive SCF (Diamond V, Cedar Rapids, IA; 21 g/d; n = 10) or no supplement (CON; n = 10) added to their diet (60% hay, 40% concentrate) for 60 d. Horses were exercised 4 d/wk for 30–45 min/d at light to moderate intensity. On d 57, horses were placed in individual stalls and tethered with their heads elevated 35 cm above wither height for 12 h to induce mild upper respiratory tract inflammation. Feces, whole blood, and nasopharyngeal flush (NPF) samples were obtained at study initiation and before and up to 72 h after stress induction to evaluate immune responses. Data were compared using mixed model ANOVA with time as a repeated measure. Prolonged head elevation resulted in greater ( P < 0.0001) serum cortisol and increases ( P < 0.05) in mucus, total leukocytes, neutrophils, lymphocytes, monocytes, and ratio of neutrophils to lymphocytes in NPF, indicative of an inflammatory response that was mostly resolved by h 72. A similar leukogram was observed in whole blood, though it was delayed 12 h compared with NPF. Neutrophils and lymphocytes were lower ( P < 0.05) and monocytes greater ( P < 0.05) in NPF from horses fed SCF than CON. Immunoglobulin (Ig) A in NPF exhibited a time × treatment effect ( P = 0.05) with an increased response to head elevation in SCF horses but no change in CON. Head elevation increased ( P < 0.05) IgA, IgG and IgM in feces, and fecal concentrations of IgA and IgG were greater ( P < 0.05) for CON than SCF. Serum IgA, IgG, and IgM were not affected by head elevation nor treatment. Changes in leukocytes and IgG in mucosal secretions suggest a stronger inflammatory response to head elevation in CON horses. Higher IgA in NPF from SCF horses may have helped attenuate local inflammation. Further study is needed to determine if SCF could improve other markers of disease resistance.

42 Feeding stalled horses a diet high omega-3 fatty acids results in a plasma fatty acid profile similar to horses on pasture

Omega-3 fatty acids (O-3) are a popular topic in equine nutrition, but it is not commonly acknowledged that a prevalent source of O-3 in a horse's diet can be from pasture. Pasture is low fat, but O-3 are the largest fraction, and horses on pasture have higher circulating O-3 than horses without pasture access. Therefore, the objective of these studies was to test the hypothesis that a feed high in O-3 that also matches the ratio of O-3:omega-6 (O-6) in pasture, would increase stalled horses’ plasma O-3 and result in a similar fatty acid profile to pastured horses. Three identical trials were completed between 2017 and 2020. Mature American Quarter Horses (n = 10 per trial; 610.5 ± 14 kg BW) were offered 0.9 kg per 454 kg BW Purina Omega Match Horse Feed (OM) and 2.0% BW Timothy hay split into equal meals (0700 a.m., 1300 p.m.) starting on d 1 of the trial. Horses were housed in individual stalls with attached dry lot turnouts for the duration of each study. The feed had a minimum of 3.5% O-3 with an O-3:O-6 ratio of 2.6. Baseline blood samples were obtained via jugular venipuncture into potassium oxalate tubes before transition to the experimental diet (d 0), and all horses were consuming a variety of other PurinaⓇ feeds before transition. Blood samples were collected on d 30 and 60 post dietary transition. Also, at d 60, blood samples were obtained from 10 horses each year at the Purina Animal Nutrition Center (Gray Summit, MO) that had free-choice access to mixed fescue pasture. These 10 horses were also fed 1.8 kg of Purina Strategy to meet nutritional needs and maintain body weight. All samples were obtained before AM feeding, were immediately centrifuged, and plasma collected for analysis. Fatty acids were analyzed via gas chromatographic-mass spectral analysis of fatty acid methyl esters. Data were analyzed using an ANOVA in SAS 9.4 (Cary, NC). All data are mean ± SD, ug/mL. Plasma O-3 levels increased from baseline (2.07 ± 0.54) to d 60 (6.29 ± 1.82) in horses consuming OM (P < 0.05). Conversely, plasma O-6 levels decreased from baseline (47.21 ± 3.86) to d 30 (42.56 ± 5.47) and remained lower at d 60 (43.89 ± 4.78) in horses consuming OM (P < 0.05). At d 60, horses consuming OM had similar plasma levels of O-3 (6.29 ± 1.82 vs 6.76 ± 2.01, P = 0.45) and O-6 (43.89 ± 4.78 vs 42.36 ± 5.80, P = 0.37) as horses on pasture, respectively. In conclusion, a feed formulated with a high level of O-3 can alter horses’ plasma O-3 concentrations until they match those of horses on pasture. This technique is helpful to boost horses’ O-3 values if they cannot tolerate pasture grass or simply do not have access to it.

97 Factors influencing horse heart rate during routine hoof care procedures

Routine hoof care procedures to remove manure, soil, and other debris packed in the underside of the hoof (PICK) and trim excess hoof growth (TRIM) are essential to a horse's health. However, these procedures may lead to acute physiological and/or psychological stress in the horse and research focusing on physiological responses of the horse to these basic procedures is limited. The objective of this study was to evaluate the influence of external factors on horse HR during routine hoof care procedures. A Polar H10 HR monitor was used to record the HR of 27 American Quarter Horses (12.1 ± 3.2 yrs; 17 mares, 7 geldings, 3 stallions) housed at The Ohio State University Equine Facility before and during routine hoof care procedures over a period of 90 d. Before each hoof care procedure, each horse was fitted with the HR monitor around the girth and allowed to acclimate to the monitor in their housing area for 5 min to establish a baseline HR. On 3 separate occasions, horses were handled by one of 2 individuals and hoof care (PICK or TRIM) was performed in either the horse's outdoor housing area or in the barn aisle by the same person for each procedure. HR was recorded every 30 s during the procedures. Data were analyzed using PROC MIXED of SAS. Avg HR increased during both hoof care procedures compared with baseline HR ( P < 0.05); however, there was no difference in avg HR between the 2 procedures. Avg HR was higher when hoof care procedures were performed in the barn aisle compared with the horse's outdoor housing area ( P < 0.0001). Avg HR was also higher when the horse was handled by the female handler compared with the male handler ( P < 0.0001). Overall, this study suggests that a horse's environment and handler may have a greater influence on horse stress than routine hoof care procedures.

33 Profiling miRNA transcripts in the equine hindgut by quantitative PCR

Gastrointestinal (GI) homeostasis in a horse results from dynamic interactions between a horse's gut physiology and the microbes in the various compartments. MicroRNAs (miRNA), single-stranded 20–22 basepair RNA molecules involved in post-transcriptional control of gene expression, have been implicated as an essential mechanism for control of gastrointestinal physiology and communication between host and microbe. Researchers have described how host-derived miRNAs influence microbial communities' composition in the GI. We have recently described how the pelvic flexure separates distinct microbial populations in the equine hindgut and subsequently have wondered if equine miRNA transcripts expressed by GI tissues could have a role in maintaining this separation. Investigations to characterize miRNAs' expression profile and other non-coding RNAs in the equine GI tract are limited. To address this, it is critical that we first know something about the expression of non-coding transcripts in the equine GI. This study investigated miRNA expression in tissues of the hindgut surrounding the pelvic flexure. RNA was isolated from the intestinal epithelium of 3 4-year-old American Quarter Horses collected from the right and left ventral colon (VC), pelvic flexure (PF), and right and left dorsal colon (DC). The miRNA transcripts were reverse transcribed using the miScript II RT kit, and relative abundance was quantified using the miScript SYBR Green PCR kit and primer sets for 286 annotated mature equine miRNAs. Biological replicates were pooled, and differential expression was determined following normalization by the delta Ct = Ct(target) – Ct(sample) method. A total of 230 miRNA transcripts were expressed in at least one GI location, with 60 expressed in all 5 locations. Twenty-eight transcripts had expression restricted to a single site, 5 of which were only expressed in the pelvic flexure. Additionally, 42 transcripts have patterns of expression across a subset of anatomical locations. Notably, 10 transcripts were expressed in the VC and PF, 5 transcripts were expressed in the DC and PF, and 15 transcripts were expressed in the VC and DC but not PF. Pathway and target analysis for the miRNAs with compartment restricted expression was performed using DIANA TOOLS. Of note, miRNA transcripts expressed only in the PF were predicted to target genes in the Hippo signaling pathway, which is associated with the regulation of intestinal tissue proliferation and homeostasis. Further investigation of miRNA expression in the equine hindgut will improve our understanding of gastrointestinal homeostasis's regulatory processes.

43 Safety of chromium propionate as a source of supplemental chromium for horses

Chromium propionate (Cr Prop) was recently approved by the US FDA for supplementation to horse feed at a level not to exceed 4 mg Cr/horse/day. This approval was based on the ability of Cr Prop to improve insulin sensitivity in adult horses following oral consumption of grain or intravenous administration of glucose, and also on the safety of Cr Prop when supplemented to horse diets at up to 2 times the FDA approved level. The objective of the current study was used to determine the safety of Cr Prop based on health, body weight (BW) gain, body condition score, and blood chemistry and hematological parameters when supplemented at levels of 0, 2, 4, or 8 mg Cr/day. Forty-eight registered Quarter horses were used in this study. The study was conducted as 4 trials, due to a limitation of individual stalls at the testing facility. Horses were housed individually and fed twice daily an oat-based concentrate mix at a rate of 0.2 kg/100 kg BW, and grass hay (2 kg/100 kg BW). Supplemental Cr was provided in 50 g of ground corn/day that was top dressed on the morning feeding of concentrate. In the fourth trial, 12 horses (n = 3/treatment) were bled before feeding on d 0 and 28 of the trial for blood chemistry and hematological measurements. Serum was analyzed for an Equine Chemistry panel, that consisted of calcium, phosphorus, magnesium, sodium, potassium, chloride, bicarbonate, osmolality, anion gap, glucose, urea, total protein, albumin, bilirubin, alkaline phosphatase, aspartate aminotransferase, and creatine kinase. A sample of whole blood also was obtained for a variety of hematological measurements. Each horse served as its own control, because d 28 blood data were statistically analyzed using d 0 blood values as a covariate. Feeding Cr Prop for 42 d did not affect health, body weight gain, or body condition score. Control horses had lower ( P ≤ 0.05) RBC counts than those fed 8 mg Cr/day, and tended to have lower RBC counts than those receiving 2 ( P = 0.07) or 4 ( P = 0.06) mg Cr/day. Other hematological measurements were not affected by dietary Cr. Serum clinical chemistry parameters measured were not affected by treatment. The present study indicates that Cr Prop is safe when supplemented to horses at up to 2 times the FDA approved feeding level.

58 Evaluation of substrate:buffer ratios for in vitro cultures inoculated with equine cecal contents

In vitro fermentation systems allow researchers to mimic the equine hindgut while providing quick, affordable, and publishable data. However, the substrate:buffer ratio within the system has a profound impact on subsequent fermentation parameters. The objective of this study was to evaluate effects of varying substrate levels on culture pH, gas production, dry matter disappearance (DMD), neutral detergent fiber disappearance (NDFD), and volatile fatty acid (VFA) concentrations utilizing the ANKOM RF Gas Production System (ANKOM Technology; Macedon, NY). Cecal fluid was collected from 4 cecally cannulated Quarter horses and used to inoculate fermentation bottles containing buffer and substrate. Substrate consisted of brome or corn included at 0.5, 1, 1.5, 2, 3, or 4 g/flask (dry matter basis) while buffer and inoculant remained constant at 125 and 25 mL/flask, respectively. Flasks were equipped with continuous gas pressure monitors and placed into a shaking incubator for 48 h at 39°C. Data were analyzed using mixed models with fixed effects of treatment, time, and treatment by time interaction, repeated effect of time, and random effects of run by horse within run. Changes in gas production, VFA production, and terminal pH were proportion to substrate amount for cultures containing 0.5 to 2 g of substrate, but were less for cultures with 3 or 4 g of substrate (linear and quadratic effects, P < 0.0001). These data suggest that cultures containing 2 g or less of substrate were substrate limited, whereas activity of cultures with more than 2 g of substrate may have been inhibited by buffer exhaustion or end product accumulation. Results of this experiment indicate that the composition of fermentative end products is impacted by substrate concentrations, and that cultures containing 125 mL of buffer and 25 mL of cecal inoculum should be limited to no more than 2 g of substrate.

95 Equine chewing is influenced by dental intervention

The mechanical breakdown of feedstuffs via mastication is the primary digestive function in the mouth, and dental abnormalities may impact the horses’ ability to adequately reduce particle size. It was hypothesized that chewing parameters would differ in horses pre- and post-dental intervention when consuming different feedstuffs. Mature Quarter horses (n = 4) and Thoroughbreds (n = 4) with an average age of 17.2 years were utilized in a trial evaluating chewing characteristics pre- and post-dental intervention. Before the start of the trial, all horses were examined by a veterinarian and assigned a dental score based on the quality of dentition. No horses utilized on the trial had received any dental intervention for 12 mo prior. The trial was separated into pre- and post-dental periods consisting of 3 weeks of data collection each. Following the final week of data collection in the pre-dental period, all horses underwent a dental floating by a veterinarian. The average dental score of horses during the pre-dental period was 3.1 indicating the presence of dental abnormalities. Following the mid-point floating, dental scores improved to an average of 2.0 indicating that abnormalities had been resolved. During each period, 5 dietary treatments (long stem hay, chopped hay, 75% Purina Ⓡ Equine Senior Ⓡ + 25% chopped hay, soaked Purina Ⓡ Equine Senior Ⓡ , and dry Purina Ⓡ Equine Senior Ⓡ ) were evaluated for 5 consecutive feedings each with horses randomly assigned a dietary treatment order in each period. Data were collected utilizing the BioNomadix EMG Device (Biopac Ⓡ Systems Inc.). Briefly, leads were attached via adhesive electrodes to both the left and right side of the horses’ jaw, corresponding to the M. Masseter muscles. Data were collected and analyzed via proprietary software to determine chew amplitude, strength, length, and rate. All dietary treatment data were analyzed via ANOVA while the pre vs. post analysis was conducted utilizing a paired Student's t -test in SAS 9.4 (Cary, NC). Chew length was longer for horses pre-dental (0.251 s) vs. post-dental (0.235 s; P < 0.05 ). Chew strength was stronger pre-dental (0.04 mV/sec) vs. post dental (0.03 mV/sec; P < 0.05 ). Horses pre-dental had a higher maximum amplitude of chews pre-dental (0.275 mV) vs. post-dental (0.235 mV; P < 0.05 ). Chew rate was lower pre-dental (1.46 chews/sec) vs. post-dental (1.54 chews/sec P < 0.05 ). Dietary treatment impacted chewing characteristics, however, no interactions existed between individual feedstuffs and chewing characteristics pre- and post-dental. These data indicate that dental intervention impacts chewing in horses, possibly allowing for more optimal mastication and breakdown of feedstuffs.

14 Evaluation of the absorption of various nutraceuticals in horses at different fed rates

Nutraceuticals are non-nutritive additives aimed at providing health benefits to the horse. Determining the efficacy of these nutraceuticals should start with an understanding of their absorption when administered through feeding. To determine the in vivo absorption of a variety of nutraceutical compounds, a trial was conducted in which various products were fed at increasing concentrations and blood samples evaluated for biomarkers. Two groups of mature Quarter Horses (n = 5 per group) were utilized in feeding trials in which compounds were fed at increasing levels over a 3 wk period. Horses were offered each amount for a period of 7 d. Horses in group 1 consumed turmeric at 3 levels (2 g/d, 20 g/d, 100 g/d), while horses in group 2 were offered hydrolyzed collagen (40 g/d, 80 g/d, 120 g/d). Following a 1 wk washout, horses in group 1 were offered green tea encapsulated in a phytosome (1 g/d, 10 g/d, 100 g/d), while horses in group 2 were offered pure green tea (0.3 g/d, 2.6 g/d, 25 g/d). Both groups received identical amounts of green tea as the phytosome form was approximately 20% green tea. Following a 1 wk washout, horses in group 1 were offered cranberry powder (10 g/d, 20 g/d, 80 g/d). All nutraceuticals were top dressed on 1.81 kg of Purina Ⓡ Omolene 200 Ⓡ twice daily at 0700 and 1500 in individual stalls. Horses were offered 1.5% BW as Timothy hay and had free-choice access to salt and water with daily turnout to drylots by group. Blood was obtained before the start of the trial and on d 1, 3, and 7 of supplementation for each compound. Serum samples were analyzed for metabolites for each compound via liquid-chromatography mass-spectrometry according to the table below. Data were analyzed using the MIXED procedure in SAS 9.4 (Cary, NC). For all dosages, turmeric biomarkers were difficult to identify in samples indicating negligible absorption. Samples from green-tea fed horses indicated that there was no difference in the phytosome vs. non-phytosome versions ( P > 0.05 ) with linear increases in serum EGCG corresponding with dosage ( P < 0.05 ). Glycine was the most consistent marker of hydrolyzed collagen absorption with a linear increase corresponding to dosage ( P < 0.05 ). Benzoic acid was only identified in samples at the 80 g/d dosage. These data are useful in developing future studies aimed at identifying the efficacy of these compounds. Utilizing these data can guide feeding amounts for further investigation.

17 Use of hair cortisol analysis to evaluate the influence of housing conditions on long-term stress in horses

Horses are kept in a variety of housing systems which often reflect the needs of both horses and their owners as well as available resources. Housing management practices not only impact horse health and performance but may also influence a horse's physiological and psychological stress. Traditional sampling methods using blood, saliva, urine, and feces are often utilized to evaluate stress in animals by measuring acute cortisol levels. Hair cortisol analysis allows for the quantification of long-term cortisol which may be a better indicator of prolonged periods of elevated stress. The objectives of this study were to evaluate the influence of transitioning horses from outdoor group housing to individual box stalls on hair cortisol concentrations (HCC) and determine the relationship between physiological and behavioral stress responses. Hair samples were collected approximately every 30 d from the same location on 11 Quarter Horses (5 mares, 6 geldings; 3.5 ± 1.3 yr) over a period of 180 d (90d group housing/90d box stalls). Hair cortisol production was measured via enzyme-linked immunosorbent assay (ELISA) and horse heart rate (HR) and behavior were recorded throughout a series of novel object and sensory sensitivity tests. Data were analyzed using PROC MIXED and Pearson correlations in SAS v. 9.4. HCC ranged from 0.6 pg/mg to 3.0 pg/mg throughout the study with an average concentration of 1.3 ± 0.4 pg/mg. Overall, there was no difference in HCC due to the type of housing system. However, HCC increased during the 30-d period immediately following the transition from group housing to individual housing and then decreased during the next 30 d period ( P < 0.05). These results suggest that the horses in this study experienced an increase in physiological stress due to the change in housing system but were able to adapt to their new environment. Interestingly, HCC were influenced by the color of the horse's mane and tail ( P < 0.01). Analysis of the relationship between physiological and behavioral responses during the novel object and sensory sensitivity tests revealed mixed results. Horse HR and reactivity were positively correlated during both sound sensitivity tests ( P < 0.05). However, behavioral observations and reactivity during the novel object, smell, and taste sensitivity tests were influenced by the stimulus ( P < 0.05). As behavioral responses may not always reflect physiological responses and only provide short-term measures of stress, hair cortisol has the potential to serve as a biomarker of long-term stress in horses and provides a tool to monitor the effects of management practices such as housing systems.

54 Evaluation of gastric health in cribbing horses fed a digestive support supplement

Cribbing is a stereotypic oral behavior whereby the horse places its incisors on a fixed object, arches its neck, pulls against the object, and emits an audible grunt. Cribbing behavior (CB) has been associated with gastrointestinal (GI) dysfunction and gastric ulceration. This randomized crossover study tested the hypothesis that dietary inclusion of a commercially available equine GI support supplement (Legends Gastrocare, Cargill, Wayzata, MN) would alter the GI environment and gastric ulcer severity in normal and cribbing horses. Mature Quarter Horses with CB (n = 4) and sex-matched non-cribbing controls (NCB; n = 4) were randomly assigned to receive either a GI support supplement (TRT) or placebo for 21 d, followed by a 2-wk washout period. Treatment groups were then switched and horses were treated for an additional 21 d. Before and after each treatment period, gastric and fecal pH were measured, and gastric ulcer severity was evaluated via endoscopic examination using an established grading system for squamous (0–4) and glandular gastric ulceration (0–1). Horses were acclimated for 2 wk before the start of the study, and throughout the study were individually stalled 16 h/d and fed bermudagrass hay (1.2% BW, DM basis) and concentrate (0.5% BW) and turned out in pairs in paddocks 8 h/d. Data were analyzed by mixed-model ANOVA with treatment, cribbing status (CB or NCB) and period included as fixed effects, and horse as a random effect. Data are presented as means ± SD. Fecal pH (6.5 ± 0.1) and gastric pH (2.9 ± 0.5) were similar between CB and NCB horses and were unaffected by TRT. Squamous mucosa ulcer scores did not differ between CB and NCB, and were unaffected by TRT. However, within CB horses, squamous ulcer severity scores were greater (indicating more severe ulceration) ( P = 0.003) with TRT (3.9 ± 0.6) than placebo (1.4 ± 0.3). Based on this pilot study, administration of a commercial GI support supplement did not alter gastric or fecal pH in normal or CB horses. Ongoing serum cortisol, serum gastrin, and microbiome analyses may help to explain the differences in gastric ulceration severity noted in CB horses when receiving TRT versus placebo. Collectively, these findings add to the growing body of literature regarding GI environment and pathophysiology in horses exhibiting CB.

Complexed trace mineral supplementation alters antioxidant activities and expression in response to trailer stress in yearling horses in training

To test the hypothesis that complexed trace mineral supplementation would increase antioxidant capacity and decrease muscle oxidative stress and damage in young horses entering an exercise training program, Quarter Horses (mean pm SD; 9.7 ± 0.7 mo) balanced by age, sex, and BW were assigned to receive complexed (CTM; n = 8) or inorganic (INORG; n = 8) trace minerals at -12 week relative to this study. Blood and muscle samples were collected before (week 0) and after 12 week of light exercise training surrounding a 1.5-h trailer stressor. Muscle glutathione peroxidase (GPx) activity was higher for CTM than INORG horses (P ≤ 0.0003) throughout the study. Following both trailer stressors, serum creatine kinase increased (P < 0.0001) and remained elevated through 24 h post-trailering (P < 0.0001). At week 0, muscle malondialdehyde, expression of superoxide dismutase 2, and whole blood GPx activity increased (Ple 0.003) following trailering but trailering did not affect these measures at week 12. Young horses supplemented with CTM had higher muscle GPx activity than horses receiving INORG, but CTM did not affect damage markers following a stressor. Dietary CTM may be useful for improving antioxidant capacity during exercise training in young equine athletes.

Intravenous formalin for treatment of haemorrhage in horses

SummaryThere is limited information in the literature regarding the clinical use of formalin for the treatment of haemorrhage in horses. This uncontrolled retrospective study summarises 11 cases that were treated with intravenous formalin at the Veterinary Health Center at Kansas State University from 2009 to 2019. The objective of this study was to describe signalment, clinical and laboratory findings, treatment and outcome of horses treated with intravenous formalin for potentially life‐threatening haemorrhagic conditions. This study does not attempt to prove efficacy of intravenous formalin, but rather to report its clinical use and the associated details of those cases. Horses ranged in age from 2 to 23 years old. There were nine Quarter Horses, one Thoroughbred and one Appaloosa. Treated conditions included haemoabdomen (4/11), uterine haemorrhage (1/11), epistaxis (3/11), haemorrhage secondary to a mandibular laceration (2/11) and haemothorax (1/11). The most utilised dose was 0.476% formalin (50 mL 10% neutral buffered formalin diluted in 1 L of isotonic fluid). Some horses were also treated with aminocaproic acid, Yunnan Baiyao, whole blood transfusions and surgery. Ten out of the 11 horses survived to discharge with one horse reported by the owner to have died 2 weeks later of an unknown cause.