Safety and efficacy of a novel iron chelator (HBED; (N,N'-Di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid)) in equine (Equus caballus) as a model for black rhinoceros (Diceros bicornis).

While iron overload disorder (IOD) and related disease states are not considered a common occurrence in domestic equids, these issues appear prevalent in black rhinoceroses under human care. In addressing IOD in black rhinos, altering dietary iron absorption and excretion may be the most globally practical approach. A main option for treatment used across other species such as humans, is chelation therapy using iron-specific synthetic compounds. As horses may serve as an appropriate digestive model for the endangered rhinoceros, we evaluated the potential use of the oral iron chelator N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED) in horses for safety and efficacy prior to testing in black rhinoceros. Health and iron digestibility and dynamics were assessed in horses (n = 6) before, and after treatment with HBED (50 mg/kg body weight) for 8 days using a crossover design with serum, faecal and urine collection. A preliminary pharmacokinetic trial was also performed but no trace of HBED was found in serially sampled plasma through 8 h post-oral dosing. HBED increased urinary iron output in horses compared to control by 0.7% of total iron intake (p < 0.01), for an average of 27 mg urinary iron/day, similar to human chelation goals. Blood chemistry, blood cell counts and overall wellness were not affected by treatment. As healthy horses are able to regulate iron absorption, the lack of change in iron balance is unsurprising. Short-term HBED administration appeared to be safely tolerated by horses, therefore it was anticipated it would also be safe to administer to black rhinos for the management of iron overload.

Sweat osmolarity shows intra-animal regional variation in the horse.

BACKGROUND: Sweating is important in regulating body temperature but can be a source of loss of both fluids and electrolytes. Although the process has been studied in horses, the variation in sweat osmolarity across the body has not. OBJECTIVES: This work describes an investigation to determine if there is regional variation in the osmolarity of sweat across different anatomical regions of the horse. ANIMALS: Ten horses were used in the study and were animals either stabled for riding lessons or had livery on-site. METHODS: Sweat samples were collected from five regions on each horse following exercise and the osmolarity measurements were made using an Osmomat 030 (Gonotec, Berlin, Germany). Values were analysed by paired t-tests and analysis of variance. RESULTS: Samples from the back and ears had statistically (P < 0.05) lower osmolarity values than those seen for the neck and forelimb, with thigh values intermediate between the other two sets of values. CONCLUSIONS AND CLINICAL IMPORTANCE: Previous studies have used osmolarity values based on the sweat collected from the horse's back. The current work demonstrates that these values are probably an underestimation of electrolyte loss, which may have implications for the composition and administration of rehydration compounds.

Investigating the gastrointestinal physiology of mature horses with and without a history of cribbing behavior in response to feeding a digestive support supplement.

Cribbing, a stereotypic oral behavior observed in horses, involves placing incisors on a fixed object, arching the neck, pulling against the object, and emitting an audible grunt. This behavior has been associated with gastrointestinal (GI) dysfunction and gastric ulceration. In this randomized crossover study, we investigated the impact of a GI support supplement (SPL) on the GI environment and physiology of four cribbing (CB) and four non-cribbing horses (NCB). Mature Quarter Horses, acclimated to individual stalls for 16 hours daily with paddock turnout in pairs for 8 hours per day, were randomly assigned to receive either the SPL or placebo for 21 days, followed by a 2-week washout period. Fecal and gastric samples were collected for pH determination and blood samples were analyzed for serum cortisol and gastrin levels. Endoscopic examinations assessed gastric ulcer severity, and cribbing frequency and bouts were recorded via video surveillance. Data were analyzed using a mixed-model ANOVA. Results showed no differences in fecal and gastric pH between cribbing statuses. However, an interaction between supplementation and cribbing status was observed for squamous mucosa ulcer scores (P=0.003). There were no differences in glandular mucosa ulcer scores, serum cortisol, serum gastrin, and crib-bite count between CB and NCB horses or between supplementation groups. Crib-bout duration did not differ with supplementation, but differences were found between periods (P<0.05) and hour ranges (P<0.001). Our findings suggest that the GI support supplement may not effectively address cribbing behavior or alter the GI environment in NCB or CB horses.

Saccharomyces cerevisiae fermentation product improves robustness of equine gut microbiome upon stress.

Introduction: Nutritional and environmental stressors can disturb the gut microbiome of horses which may ultimately decrease their health and performance. We hypothesized that supplementation with a yeast-derived postbiotic (Saccharomyces cerevisiae fermentation product-SCFP) would benefit horses undergoing an established model of stress due to prolonged transportation. Methods: Quarter horses (n = 20) were blocked based on sex, age (22 ± 3 mo) and body weight (439 ± 3 kg) and randomized to receive either a basal diet of 60% hay and 40% concentrate (CON) or the basal diet supplemented with 21 g/d Diamond V TruEquine C (SCFP; Diamond V, Cedar Rapids, IA) for 60 days. On day 57, horses were tethered with their heads elevated 35cm above wither height for 12 h to induce mild upper respiratory tract inflammation. Fecal samples were collected at days 0, 28, and 56 before induction of stress, and at 0, 12, 24, and 72 h post-stress and subjected to DNA extraction and Nanopore shotgun metagenomics. Within sample (alpha) diversity was evaluated by fitting a linear model and between sample (beta) diversity was tested with permutational ANOVA. Results: The SCFP stabilized alpha diversity across all time points, whereas CON horses had more fluctuation (P < 0.05) at 12, 24, and 72 h post-challenge compared to d 56. A significant difference between CON and SCFP was observed at 0 and 12 h. There was no difference in beta-diversity between SCFP and CON on d 56. Discussion: Taken together, these observations led us to conclude that treatment with SCFP resulted in more robust and stable microbial profiles in horses after stress challenge.

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.

Digestibility and nitrogen and water balance in horses fed rhizoma peanut hay.

Rhizoma peanut (RP, Arachis glabrata) hay has the potential to meet horses' crude protein (CP) requirements with less nitrogen excretion than other legumes. This study aimed to evaluate nutrient intake, apparent digestibility, and nitrogen balance of RP "Florigraze" hay compared with alfalfa (ALF, Medicago sativa L. "Legendary XHD") and bermudagrass (BG, Cynodon dactylon L. "Coastal") hays when fed to maintenance horses at 2% body weight/d on a dry matter (DM) basis. We hypothesized that nutrient intake would be comparable between the legume hays and lesser for BG and that RP would result in reduced nitrogen excretion compared with ALF. Six mature Quarter Horse geldings (593 ± 40 kg; mean ± SD) were randomly assigned to one of the hays in a replicated 3 × 3 Latin square with 21-d periods. A 14-d adaptation phase was followed by a 3-d total fecal and urine collection. Days 18 to 21 were used for a companion study. Intake of nutrients is reported on a DM basis. Digestible energy (DE) intakes from ALF (29.91 Mcal/d) and RP (29.37 Mcal/d) were greater (P < 0.0001) than BG (20.78 Mcal/d). CP intake was greater (P < 0.0001) for ALF (2.5 kg/d), followed by RP (1.9 kg/d) and BG (1.5 kg/d). All hays exceeded maintenance requirements for DE, CP, Ca, and P. Apparent digestibility of DM and CP was greatest (P < 0.0001) for ALF (69% and 84%), intermediate for RP (61% and 72%), and least for BG (46% and 64%). Apparent digestibility of neutral detergent fiber did not differ (P = 0.2228) among hays, while digestibility of acid detergent fiber (P = 0.0054) was least for RP but similar for ALF and BG. Water intake (kg/d) for ALF (57) was greater (P = 0.0068) than RP (45) and BG (41). Greater (P = 0.0271) water retention (kg/d) was observed for ALF (13.5), followed by RP (10.8) and BG (7.5). There was a difference in nitrogen excretion, with greatest urinary nitrogen excretion for ALF (P < 0.0001) and greatest fecal nitrogen excretion for BG (P = 0.0001). Total nitrogen excretion was greater (P < 0.0001) for ALF (278 g/d), followed by RP (211 g/d) and BG (179 g/d). Nitrogen retention was greater (P = 0.0005) for ALF when represented as g/d (ALF: 129, RP: 86, and BG: 57 g/d) but similar (P = 0.0377) to RP when presented as percent of nitrogen intake (ALF: 32%, RP: 29%, and BG: 24%). Results indicate that RP hay is a suitable legume for horses by meeting DE and CP requirements and having a significant reduction in nitrogen compared with ALF.

Characterization of Forage Utilization and Pasture Management Practices on Florida Horse Operations.

This study used an online survey to gather information on forage utilization and pasture management practices implemented on horse farms in Florida. We hypothesized that pastures were inadequately managed and underutilized, resulting in greater reliance on hay. Data from 80 complete responses were submitted to descriptive analysis and Fisher's exact test. Significantly associated variables were also evaluated using multiple correspondence analysis. The primary function of the facility most reported was recreational horse use (62%). Nearly all operations (n = 79) had pasture available, with 57% reporting housing horses on pasture full time (24 hr/d) and 48% reporting implementation of rotational stocking. Most respondents reported using a combination of hay and pasture (57.5%) as their primary source of forage, followed by pasture only (32.5%) and hay only (10%). Pasture maintenance was performed by 82% of respondents, and 6% did not implement any type of pasture management. Primary forage fed to the horses was related to the type of pasture management implemented (P = .0313) and turnout practices (P = .0003). The multiple correspondence analysis showed that relying on hay as the primary source of forage was associated with a shorter duration of pasture access and lack of pasture management. Most operations did not have their hay (89%) or pasture (94%) analyzed for nutrient composition. Despite pasture availability, lack of adequate pasture management appeared to contribute to a greater dependence on hay feeding. Pasture management practices and forage analysis are potential areas for horse owner education in Florida.

Postprandial Blood Glucose and Insulin Responses of Horses to Feeds Differing in Soluble Fiber Concentration.

In humans, the consumption of soluble fibers reduces glycemic response after a meal. We hypothesized high soluble fiber diets would reduce and delay postprandial glucose and insulin responses in horses. In a 4 × 4 Latin square design experiment, four Quarter Horse geldings were adapted to diets containing orchardgrass hay (ORCH) or ORCH with 1 of 3 treatment ingredients: molassed sugar beet pulp (BEET), almond hulls (HULL), or steam-crimped oats (OATS). Blood was serially sampled for 6 hours after feeding 0.15% body weight (BW) of the treatment ingredient (meal test) or 1.1 g starch/kg BW from oats plus the treatment ingredient (starch test) to evaluate glycemic and insulinemic responses. Glycemic response during the meal test peaked between 60 and 90 min after feeding (P < .05) and tended to be altered by diet (P = .071) and diet × time (P = .076). Serum insulin was affected by diet (P = .008), time (P < .001), and diet × time (P < .001) during the meal test, with concentrations lower in ORCH compared with BEET and OATS (P < .05). In the starch test, glucose was lower (P < .05) in ORCH and HULL compared with BEET and insulin was lower (P = .046) in ORCH compared with BEET. In both tests, horses took longer (P < .05) to consume HULL, likely influencing postprandial responses. Future research integrating the functional properties of feeds with physiological responses will be necessary to elucidate how soluble fiber affects postprandial glucose metabolism in horses.

A Pilot Study Exploring the Relationship Between Digesta Retention Time in the Equine Gastrointestinal Tract and Compartment Models.

Digesta retention time within specific segments of the equine gastrointestinal tract (GIT) may be more relevant to scientific inquiries than total tract mean retention time (TTMRT); however, measuring retention time in individual segments requires access to the digestive tract. The objective of this study was to compare prececal, cecal, and colonic mean retention time (MRT) with model-derived compartment MRT. A cecally fistulated gelding was fed indigestible particulate and liquid markers to determine TTMRT and dosed with different pairs of particulate and liquid markers into the cecum (hindgut MRT) and into the right ventral colon through the cecocolic orifice (colon MRT). Fecal marker concentrations were fit to stochastic and mechanistic models using nonlinear least squares methods (MATLAB). Total tract MRT and MRT for each GIT segment were compared with model-derived compartment MRT using paired t-test to determine differences and two one-sided tests to determine equivalence. All models resulted in parameter estimates and an acceptable fit to fecal marker excretion curves, but some parameter estimates did not differ from zero (95% CI included 0). Model-derived TTMRT were equivalent (P < .05) to arithmetically calculated MRT. Most GIT segment MRT differed (P < .05) from model-derived compartment MRT. Differences ranged from -26.1 hours to 25.8 hours. In these exploratory data, model derived compartment retention times failed to pair with MRT in different GIT segments. Significant methodological and analytical challenges remain to describe retention time in individual segments of the equine GIT.

Digestibility and Retention Time of Coastal Bermudagrass (Cynodon dactylon) Hay by Horses.

Bermudagrass (Cynodon dactylon) and other warm-season grasses are known for their increased fiber concentrations and reduced digestibility relative to cool-season grasses and legumes. This study investigated the digestive characteristics and passage kinetics of three maturities of Coastal bermudagrass hay. A 5 × 5 Latin square design experiment was used to compare the digestion of five hays: alfalfa (Medicago sativa, ALF), orchardgrass (Dactylis glomerata, ORCH), and Coastal bermudagrass harvested at 4 (CB 4), 6 (CB 6), and 8 weeks of regrowth (CB 8). Horses were fed cobalt-ethylenediaminetetraacetic acid (Co-EDTA) and ytterbium (Yb) labeled neutral detergent fiber (NDF) before an 84-h total fecal collection to determine digesta retention time. Dry matter digestibility was greatest for ALF (62.1%) and least for CB 6 (36.0%) and CB 8 diets (36.8%, SEM = 2.1; p < 0.05). Mean retention time was longer (p < 0.05) for Coastal bermudagrass (particulate 31.3 h, liquid 25.3 h) compared with ORCH and ALF (28.0 h, SEM = 0.88 h; 20.7 h, SEM = 0.70 h). Further evaluation of digesta passage kinetics through mathematical modeling indicated ALF had distinct parameters compared to the other diets. Differences in digestive variables between forage types are likely a consequence of fiber physiochemical properties, warranting further investigation on forage fiber and digestive health.

Skeletal muscle from aged American Quarter Horses shows impairments in mitochondrial biogenesis and expression of autophagy markers.

Aging is associated with decreased mitochondrial content and function in skeletal muscle, possibly due to compromised biogenesis and autophagic removal of dysfunctional mitochondria. The aim of this study was to compare markers of mitochondrial content and biogenesis and of autophagy between skeletal muscle from young and aged American Quarter Horses. Citrate synthase protein and mtDNA copy number were decreased in triceps brachii (TB) muscle (P<0.05) from aged horses, suggesting an age-related decline in mitochondrial content. Concomitantly, mRNA expression of PGC-1α and TFAM, regulators of mitochondrial biogenesis, was lower in aged compared to young TB (P<0.05). Expression of autophagy markers suggested an age-associated decline of autophagy. The autophagosomal cargo protein SQSTM/p62 accumulated with age in both muscles (P<0.05). Expression of Autophagy-related protein Atg5 (P<0.05) and the autophagosome-bound form of Microtubule-associated protein light chain 3 (LC3-II; P<0.05) were lower in aged compared to young TB. While LC3 transcript level was elevated in aged compared to young GM (P<0.001), protein expression of LC3-II was unaffected. Gene expression of Lysosomal Membrane-Associated Protein 2 (LAMP2) was not affected by age in either muscle. However, LAMP2 protein expression declined with age (P<0.05), suggesting a decline in autophagosome-lysosome fusion. Taken together, our data indicate that equine skeletal muscle mitochondrial content and biogenesis were impaired with age. Further, autophagosome formation and lysosomal degradation were negatively affected in aged TB and GM, respectively. Future research needs to explore whether interventions targeting these cellular processes can prolong health and performance of aging American Quarter Horses.

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 (P CI , P CI+II ; P = 0.008) and electron transport capacity (E CI+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 P CI , P CI+II , E CI+II , and FCFCI and FCF CI+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.

Effects of aging on mitochondrial function in skeletal muscle of American American Quarter Horses.

Skeletal muscle function, aerobic capacity, and mitochondrial (Mt) function have been found to decline with age in humans and rodents. However, not much is known about age-related changes in Mt function in equine skeletal muscle. Here, we compared fiber-type composition and Mt function in gluteus medius and triceps brachii muscle between young (age 1.8 ± 0.1 yr, n = 24) and aged (age 17-25 yr, n = 10) American Quarter Horses. The percentage of myosin heavy chain (MHC) IIX was lower in aged compared with young muscles (gluteus, P = 0.092; triceps, P = 0.012), while the percentages of MHC I (gluteus; P < 0.001) and MHC IIA (triceps; P = 0.023) were increased. Mass-specific Mt density, indicated by citrate synthase activity, was unaffected by age in gluteus, but decreased in aged triceps (P = 0.023). Cytochrome-c oxidase (COX) activity per milligram tissue and per Mt unit decreased with age in gluteus (P < 0.001 for both) and triceps (P < 0.001 and P = 0.003, respectively). Activity of 3-hydroxyacyl-CoA dehydrogenase per milligram tissue was unaffected by age, but increased per Mt unit in aged gluteus and triceps (P = 0.023 and P < 0.001, respectively). Mt respiration of permeabilized muscle fibers per milligram tissue was unaffected by age in both muscles. Main effects of age appeared when respiration was normalized to Mt content, with increases in LEAK, oxidative phosphorylation capacity, and electron transport system capacity (P = 0.038, P = 0.045, and P = 0.007, respectively), independent of muscle. In conclusion, equine skeletal muscle aging was accompanied by a shift in fiber-type composition, decrease in Mt density and COX activity, but preserved Mt respiratory function.

Influence of L-arginine supplementation on reproductive blood flow and embryo recovery rates in mares.

Supplementation with L-arginine can increase uterine arterial blood flow and vascular perfusion of the preovulatory follicle in mares. Increased vascular perfusion of the preovulatory follicle has been correlated with successful pregnancy in mares. The objective of this study was to determine if supplemental L-arginine would increase ovarian arterial blood flow, vascular perfusion of the preovulatory follicle, and embryo recovery rates in mares. Mares were blocked by age and breed and assigned at random within block to L-arginine supplementation or control groups. Mares were fed L-arginine beginning 17 days before and through the duration of the study. Transrectal Doppler ultrasonography was used to measure ovarian arterial blood flow and vascular perfusion of the preovulatory follicle daily when it reached 35 mm and subsequent CL on Days 2, 4, and 6. Mares, on achieving a follicle of 35 mm or more were bred via artificial insemination and an embryo collection was attempted 7 days after ovulation. Treatment did not affect interovulatory interval (arginine-treated, 18.1 ± 2.6 days; control, 20.7 ± 2.3 days) or embryo recovery rate (arginine-treated, 54%; control, 48%). Mares treated with l-arginine had a larger follicle for the 10 days preceding ovulation than control mares (30.4 ± 1.2 and 26.3 ± 1.3 mm, respectively; P < 0.05) and vascular perfusion of the dominant follicle tended (P = 0.10) to be greater for the 4 days before ovulation. No differences were observed between groups in diameter or vascular perfusion of the CL. Resistance indices, normalized to ovulation, were not significantly different between groups during the follicular or luteal phase. Oral l-arginine supplementation increased the size and tended to increase perfusion of the follicle 1, but had no effect on luteal perfusion or embryo recovery rates in mares.

Oral L-arginine supplementation impacts several reproductive parameters during the postpartum period in mares.

L-arginine is an amino acid which can alter pituitary function and increase blood flow to the reproductive tract. The objective was to determine the effect of supplementing 100g of L-arginine on plasma arginine concentrations, follicular dynamics and ovarian and uterine artery blood flow during the estrus that occurs subsequent to foaling. In Experiment 1, mares were fed 100g L-arginine for 1 day during the last 3 weeks of pregnancy and plasma samples taken for every hour for the first 4h and every other hour until 12h.L-arginine supplementation elevated plasma arginine concentrations from 1 to 8h post feeding; arginine peaked at 6h (arginine: 515±33µmol/L; control: 80±33µmol/L). In Experiment 2, mares received either 100g L-arginine or control diets beginning 21 d before the expected foaling date and continued for 30 d postpartum. The reproductive tract was evaluated by transrectal Doppler ultrasonography from Day 1 postpartum through Day 30. There were no differences in ovarian follicular dynamics, ovarian or uterine resistance indices between groups. Vascular perfusion of the F1 follicular wall was greater in L-arginine supplemented mares (37.3±2.6%) than controls (25.4±2.7%; P<0.05). L-arginine supplemented mares had a smaller uterine body and horns and accumulated less uterine fluid than controls (P<0.05). The combination of reducing uterine fluid accumulation, while not altering follicular development, raises the possible use of L-arginine supplementation as a breeding management tool during the postpartum period to increase reproductive success.