Time-Budget and Welfare Indicators of Stabled Horses in Three Different Stall Architectures: A Cross-Sectional Study.

Keeping horses in single stalls can lead to the development of abnormal and stereotypic behaviors (ASB). Opportunities for social interactions and stall architecture can influence behavior. The current study aimed to identify how three different stall architectures influenced time-budget and physiological parameters in horses. Stall types included: (1) 3.2 × 3.7 m with tactile contact between horses (B1); (2) 2.6 × 3.5 m with visual contact between horses and outside view (B2); (3) 2.3 × 3.4 m with visual contact and outside view (B3). Ten horses from B1 and B3, and nine from B2 were randomly selected, filmed for 24 hours and the video was analyzed with continuous behavioral sampling. Nine horses from B1, 8 from B2 and 7 from B3 were randomly selected for blood sampling used to determine cortisol levels, cortisol circadian rhythm (CCR), white blood cells (WBC) and neutrophil/lymphocyte ratio. The effects of different stall architectures were analyzed by ANOVA and post hoc Tukey test. B1 had higher social interaction time (2.77% of the time-budget) (P = .020), with no other differences. Time spent eating represented a low proportion of horses' time-budget (14.31%) and all horses demonstrated ASB behaviors (21.10% of the time-budget). Twenty-five percent of the horses presented low WBC, 38% of the horses had high cortisol levels, and 29% of the horses had an altered CCR. Those alterations along with high prevalence of ASB indicate that horses were in a state of chronic stress. The stalls' architecture did not affect the presence of abnormal behaviors or indicators of stress.

Molecular and Cellular Evaluation of Horses With Summer Pasture Associated Asthma Syndrome.

Equine asthma is an airway disease that affects a large number of horses annually leading to considerable economic losses in the horse industry. Despite advances in research in this area, there is still a lack of information on its etiology and molecular characterization in pasture associated asthma. The objective of the current study was to characterize the inflammatory disease of lower airways in horses maintained on pasture through cytologic and immunologic profile during the summer in a tropical environment by analysis of the gene expression of Th1 cytokines (IFN- λ, IL-8), Th2 cytokines (IL-4 and IL-5), and pro-inflammatory cytokines (IL-1, TNF-α) in the bronchoalveolar lavage (BAL) fluid in healthy and asthma horses on pasture. A group 39 of clinically healthy horses maintained on native pasture and supplemented with concentrate was evaluated by BAL analyzed for differential cellular count and assigned into a control and an asthma group. The gene expression of pro-inflammatory cytokines was analyzed in the BAL by reverse time PCR (RT-PCR) (IL-1α (alpha), IL-4, IL-5, IL-8, TNF-α alpha and IFN-λ), using ß-actin as housekeeping gene. Higher gene expression of IL-1, IL-4, IL-5, IL-8, IFN-λ in the BAL of asthma horses was found. Current results indicate an increase in Th2, characterizing an allergic inflammatory reaction due to the significant increase in IL-5 in asthmatic horses (10.3 ± 1.13), when compared to the values ​​obtained in normal horses (3.27 ± 0.46). The only down regulated cytokine in the asthma group was TNF-α, suggesting a chronic antigenic reaction.

Effects of Different Hay Feeders, Availability of Roughage on Abnormal Behaviors and Cortisol Circadian Rhythm in Horses Kept in Dry Lots.

Free choice forage could be the best option regarding horses' welfare but can lead to increased body weight (BW), and waste of hay. Automatic box feeders (BF) and slow feeders (SF) decrease food waste, but it is unknown how these affect the horses' time-budget (TB). This study compared the effects of feeding free choice hay (FC), to a SF and an automated BF on the horses' cortisol circadian rhythm (CCR) and behavior by 24-hours continuous behavioral sampling (CBS). The study was designed as a 3 × 3 Latin square design with 15 polo horses divided into 3 groups, for 15 days on each treatment. Every 15 days, BW was assessed, blood collected for CCR analysis, the behavior recorded during the last 24 hours of the last day of each treatment and the video analyzed with CBS. Time spent on all behaviors was evaluated and used for the determination of the animals' TB. The effects of the different feeders were analyzed with ANOVA. FC horses consumed and wasted more hay daily (16.6 ± 0.5kg) (P < .001), compared with BF (10.4 ± 0.5 kg), and SF (9.30 ± 0.45 kg). FC horses had higher weight gain (P < .001, 23.5 ± 4.6kg), compared to BF (1.2 ± 5.7 kg) and SF (0.37 ± 4.6) kg. FC and SF horses spent more than 50% of the TB foraging, generating a TB similar to grazing horses. BF horses spent less time eating (P < .001), increasing time spent standing, sniffing the ground, and practicing coprophagy (P < .050). BF horses showed the highest aggression (P < .043). CCR was not different among treatments.

Effects of corn supplementation on serum and muscle microRNA profiles in horses.

Laminitis associated with equine metabolic syndrome causes significant economic losses in the equine industry. Diets high in non-structural carbohydrates (NSC) have been linked to insulin resistance and laminitis in horses. Nutrigenomic studies analyzing the interaction of diets high in NSCs and gene expression regulating endogenous microRNAs (miRNA) are rare. This study's objectives were to determine whether miRNAs from dietary corn can be detected in equine serum and muscle and its impacts on endogenous miRNA. Twelve mares were blocked by age, body condition score, and weight and assigned to a control (mixed legume grass hay diet) and a mixed legume hay diet supplemented with corn. Muscle biopsies and serum were collected on Days 0 and 28. Transcript abundances were analyzed using qRT-PCR for three plant-specific and 277 endogenous equine miRNAs. Plant miRNAs were found in serum and skeletal muscle samples with a treatment effect (p < .05) with corn-specific miRNA being higher than control in serum after feeding. Endogenous miRNAs showed 12 different (p < .05) miRNAs in equine serum after corn supplementation, six (eca-mir16, -4863p, -4865p, -126-3p, -296, and -192) previously linked to obesity or metabolic disease. The results of our study indicate that dietary plant miRNAs can appear in circulation and tissues and may regulate endogenous genes.

Comparison of an Antioxidant Source and Antioxidant Plus BCAA on Athletic Performance and Post Exercise Recovery of Horses.

Antioxidant supplementation decreases postexercise oxidative stress but could also decrease muscle protein synthesis. This study compared the effects of three diets: low antioxidant (control, CON), high antioxidant (AO), and branched-chain amino acid high antioxidant (BCAO) supplementation on postexercise protein synthesis and oxidative stress. We hypothesized that supplementing antioxidants with branched-chain amino acids(BCAA) would reduce oxidative stress without hindering muscle protein synthesis. Eighteen mixed-breed polo horses (11 mares and 7 geldings, with age range between 5 and 18 years, were on CON diet for 30 days (from day -45 until day 0) and then were assigned to one of the treatments after the first lactate threshold test (day 0, LT). LT were also conducted on days 15 and 30 of supplemenation. Oxidative stress was assessed by measuring blood glutathione peroxidase, superoxide dismutase, and malondialdehyde concentrations before 2 and 4 hours after each LT. Muscle biopsies were taken before and 4 hours after each LT and analyzed for gene expression of protein synthesis by RTqPCR. Data were analyzed by ANOVA and compared by least-square means. A reduction in oxidative stress occurred over time (P < .05), from day 0 to day 30. An up-regulation in the abundance of muscle protein mRNA transcripts was found for CD36, CPT1, PDK4, MYF5, and MYOG (P < .05) after all lactate threshold tests, without a treatment effect. A treatment-by-exercise effect was observed for MYOD1 (P = .0041). Transcript abundance was upregulated in AO samples post exercise compared to other treatments. MYF6 exhibited a time-by-treatment effect (P = .045), where abundance increased more in AO samples from day 0 to day 15 and 30 compared to other treatments. Transcript abundance for metabolic and myogenic genes was upregulated in post exercise muscle samples with no advantage from supplementation of antioxidants with branched-chain amino acids compared to antioxidants alone.

Equine Fecal Microbiota Changes Associated With Anthelmintic Administration.

The gastrointestinal microbiota (GIM) plays an essential role in maintaining intestinal homeostasis with disruptions having profound effects on the wellbeing of the host animal. Parasitic infection is a long-standing issue for the equine industry, and the use of anthelmintic drugs for parasite control has long been standard practice. The impact of anthelmintic treatment on the GIM in healthy horses is not well known. This study evaluated the hypothesis that anthelmintic administration will alter the equine fecal microbiota in horses without an observed helminth infection. Ten horses were treated with a single dose of QUEST PLUS (active ingredients: Moxidectin and Praziquantel) (Zoetis), and fecal samples were collected before and after treatment. Amplicon sequencing data were quality filtered, processed, and analyzed using QIIME2. Anthelmintic treatment corresponded with a small but significant decrease in alpha diversity (P-value < .05). Analysis of taxonomic abundances before and after treatment with DESeq2 identified 21 features that were significantly different after treatment (Padj-value < .05). Differences in beta diversity associated with treatment were not significant and potentially suggest factors unique to the individual may play an essential role in the specific responses observed. Overall, the present study does not indicate a broad, large-scale impact on the GIM after anthelmintic treatment. The results do, however, suggest the potential of individualized responses that are based instead on host factors. Identification of these factors and investigation of their impact on the host/microbiota relationship will contribute significantly to our understanding of the role of the microbiome in horse health.

The Effects of Various Levels of Docosahexaenoic Acid on Inflammatory Markers in Conditioned Horses During Lactate Threshold Tests.

Exercise stimulates the release of inflammatory cytokines and supplementation with n-3 fatty acids reduces inflammation. The effects of different doses of docosahexaenoic acid (DHA) on inflammation in polo horses submitted to field lactate threshold tests (LT) were analyzed. We hypothesized that higher doses of DHA would reduce postexercise inflammation. Twenty polo horses were assigned to different treatments: control group fed (n = 5) grain and hay, 3 treatment groups (n = 5) fed 10, 20, or 50 g/day of DHA with grain and free choice hay during 60 days. Horses underwent LT tests before start, 30, and 60 days of supplementation. Blood samples were taken at rest for blood cytokine expression (CEx), plasma cytokine enzyme-linked immunosorbent assay (CEL), fatty acid, vitamin E, and creatine kinase (CK) analysis, after LT for CEx analysis (interferon gamma, tumor necrosis factor alpha [TNF-α], interleukin-1 [IL-1], interleukin-6 [IL-6], interleukin-10 [IL-10]), CEL, and CK analysis. Effects of treatment, time, and exercise were analyzed by analysis of variance, significant results compared by least square means analysis, and significance set at P < .05. There was a dose-dependent increase in plasma DHA, and highest arachidonic acid was found in 20 and 50 g. Vitamin E was lowest in 20 and 50 g. LT did not change IL-6, downregulated IL-1 and TNF-α, upregulated IL-10, and interferon gamma. The 10 g led to postexercise downregulation of interferon gamma and IL-10 CEx compared to other treatments. A lack of antioxidants in the supplements may have led to the absence of treatment effects in the 20 and 50 g. 10 g DHA helped moderate postexercise inflammation.

Effects of Feeding Frequency Using a Commercial Automated Feeding Device on Gastric Ulceration in Exercised Quarter Horses.

The objective of this study was to evaluate two feeding regimens: traditional grain feeding (twice a day, traditional fed [TF]) compared with horses fed with an automated feeder programmed to deliver grain in 20 equal aliquots (fractioned fed [FF]) and its effects on gastric ulceration, body weight, and body condition score (BCS). Thirty-one Quarter Horses were maintained in individual stalls during the 60-day experimental period and worked at moderate exercise intensity. Gastroscopies were performed before grain adaptation at 0, 30, and 60 days. Weight and BCS were evaluated biweekly. There were no changes in squamous (equine squamous gastric disease [ESGD]) ulcers in FF horses over the study period and higher ESGD ulcer scores in TF horses over both the 30 and 60-day time points. The proportion of horses with equine glandular gastric disease (EGGD) were significantly lower with fractioned feed when compared with traditional feed on day 30, but no difference was seen at day 60. No significant differences were found when comparing the two groups for BCS or body weight throughout the study period. This study further supports that fasting period is a strong contributing factor for gastric ulcer formation. FF of the daily total grain led to a decrease in time fasting when compared with TF which reduced the severity and incidence of both ESGD and EGGD. The use of the commercial feeder for FF may aid in lessening prevalence of gastric ulcers in young training Quarter Horses.

Fructokinase, Fructans, Intestinal Permeability, and Metabolic Syndrome: An Equine Connection?

Fructose is a simple sugar present in honey and fruit, but can also exist as a polymer (fructans) in pasture grasses. Mammals are unable to metabolize fructans, but certain gram positive bacteria contain fructanases and can convert fructans to fructose in the gut. Recent studies suggest that fructose generated from bacteria, or directly obtained from the diet, can induce both increased intestinal permeability and features of metabolic syndrome, especially the development of insulin resistance. The development of insulin resistance is driven in part by the metabolism of fructose by fructokinase C in the liver, which results in oxidative stress in the hepatocyte. Similarly, the metabolism of fructose in the small bowel by intestinal fructokinase may lead to increased intestinal permeability and endotoxemia. While speculative, these observations raise the possibility that the mechanism by which fructans induce laminitis could involve intestinal and hepatic fructokinase. Further studies are indicated to determine the role of fructanases, fructose and fructokinase in equine metabolic syndrome and laminitis.

Dietary energy source affects glucose kinetics in trained Arabian geldings at rest and during endurance exercise.

Advances in modeling and tracer techniques provide new perspective into glucose utilization and potential consequences to health or exercise performance. This study used stable isotope and compartmental modeling to evaluate how adaptation to a feed high in sugar and starch (SS) compared with a feed high in fat and fiber (FF) affects glucose kinetics at rest and during exercise in horses. Six trained Arabians adapted to each feed underwent similar tests at rest and while running approximately 4 m/s on a treadmill. For both tests, horses received 100 micromol/kg body weight [6,6-(2)H]glucose through a venous catheter. Circulating tracer glucose was described for 150 min by exponential decay curves and compartmental analysis. All parameters of glucose transfer increased with exercise (P < or = 0.004). Compared with FF horses, SS horses had higher circulating glucose (P = 0.022) and fractional glucose transfer rates (min(-1)) at rest (P = 0.055). Exercise increased glucose irreversible loss (mmol/min) more in SS horses (P = 0.037). Total glucose transfer during exercise tended to be greater in SS horses (0.027 +/- 0.002 mmol/min) compared with FF horses (0.023 +/- 0.002 mmol/min) (P = 0.109). This study characterized the effect of diet on glucose kinetics in resting and exercising horses using new modeling methods. Horses adapted to a fat-supplemented feed utilized less glucose during low-intensity exercise. Fat supplementation in horses may therefore promote greater flexibility in the selection of substrate to meet energy demands for optimal health and performance.

Mammary adenocarcinoma in a mare

A non-lactating 17-year-old grey barren Mangalarga Marchador mare was referred to the Large Animal Veterinary Hospital of the Federal Rural University of Rio de Janeiro (UFRRJ), presenting enlargement of the mammary gland. The owner reported that the animal had a lesion in the mammary gland for at least two years, diagnosed and treated as chronic mastitis. Initially only the right gland was involved, presenting ulceration and exudation on the cutaneous surface. After 20 months, the left gland also became affected. The diagnosis of tubulo-papillary adenocarcinoma was based on the typical microscopic lesions. This work shows the importance of the histopathological examination in the differential diagnosis between the neoplasms and the chronic inflammation in the mammary gland of mares, as well as, to show that the cytological examination cannot detect the tumor, in case the puncture is made in areas of secondary infection.

Uma égua tordilha, Mangalarga Marchador, de 17 anos, não-lactante e não-prenhe, foi encaminhada ao Hospital de Grandes Animais da Universidade Federal Rural do Rio de Janeiro, com histórico de mastite crônica há dois anos. No início da lesão, apenas a glândula mamária direita estava muito aumentada, com exsudação sero-hemorrágica e ulcerada. Após 20 meses, a mama esquerda também estava comprometida. A égua foi tratada para mastite crônica, porém o exame histopatológico revelou tratar-se de um adenocarcinoma túbulo-papilar. Este trabalho evidencia a importância do exame histopatológico no diagnóstico diferencial entre neoplasias e inflamação crônica da mama de éguas, uma vez que o exame citopatológico pode não detectar o tumor, quando a punção é feita em áreas de infecção secundária.

10.1093/jn/136.7.2090S

Treatment of clinical laminitis usually fails to prevent some degree of persistent disability; thus, intervention should aim at avoiding risk factors and preventing the disease. Efficiency of intervention would be improved by identifying predisposed horses and ponies. A herd of 160 healthy ponies included 54 previously laminitic (PL) and 106 never laminitic (NL). Pedigree analysis was consistent with dominant inheritance partially suppressed in males. Blood analysis revealed higher plasma concentrations of insulin and triglycerides but not cortisol, glucose, or free fatty acids in the PL group. Proxies for insulin sensitivity and beta-cell responsiveness, which were calculated from plasma insulin and glucose, indicated compensated insulin resistance in the PL group. A prelaminitic metabolic syndrome (PLMS) was derived statistically to have cut-off points for the 2 proxies, hypertriglyceridemia, and body condition score. It had a total predictive power of 78%. It identified 62 ponies with PLMS, and 98 as PLMS-free. Two months later, pasture starch concentration doubled, and 13 clinical cases of laminitis developed, 11 in the PLMS group and 2 in the PLMS-free group, giving an odds ratio of 10.4 (P = 0.0006). The PLMS can be used to identify predisposed ponies in need of special care; the efficiency of intervention would increase nearly 3-fold in the present case. It enables the design of new interventions suitable for testing. The PLMS also might influence market values.

Evaluation of genetic and metabolic predispositions and nutritional risk factors for pasture-associated laminitis in ponies.

OBJECTIVE: To evaluate genetic and metabolic predispositions and nutritional risk factors for development of pasture-associated laminitis in ponies. DESIGN: Observational cohort study. ANIMALS: 160 ponies. PROCEDURES: A previous diagnosis of laminitis was used to differentiate 54 ponies (PL group) from 106 nonlaminitic ponies (NL group). Pedigree analysis was used to determine a mode of inheritance for ponies with a previous diagnosis of laminitis. In early March, ponies were weighed and scored for body condition and basal venous blood samples were obtained. Plasma was analyzed for glucose, insulin, triglycerides, nonesterified fatty acids, and cortisol concentrations. Basal proxies for insulin sensitivity (reciprocal of the square root of insulin [RISQI]) and insulin secretory response (modified insulin-to-glucose ratio [MIRG]) were calculated. Observations were repeated in May, when some ponies had signs of clinical laminitis. RESULTS: A previous diagnosis of laminitis was consistent with the expected inheritance of a dominant major gene or genes with reduced penetrance. A prelaminitic metabolic profile was defined on the basis of body condition, plasma triglyceride concentration, RISQI, and MIRG. Meeting > or = 3 of these criteria differentiated PL- from NL-group ponies with a total predictive power of 78%. Determination of prelaminitic metabolic syndrome in March predicted 11 of 13 cases of clinical laminitis observed in May when pasture starch concentration was high. CONCLUSIONS AND CLINICAL RELEVANCE: Prelaminitic metabolic syndrome in apparently healthy ponies is comparable to metabolic syndromes in humans and is the first such set of risk factors to be supported by data in equids. Prelaminitic metabolic syndrome identifies ponies requiring special management, such as avoiding high starch intake that exacerbates insulin resistance.

Use of proxies and reference quintiles obtained from minimal model analysis for determination of insulin sensitivity and pancreatic beta-cell responsiveness in horses.

OBJECTIVE: To develop proxies calculated from basal plasma glucose and insulin concentrations that predict insulin sensitivity (SI; L.min(-1) x mU(-1)) and beta-cell responsiveness (ie, acute insulin response to glucose [AIRg]; mU/L x min(-1)) and to determine reference quintiles for these and minimal model variables. ANIMALS: 1 laminitic pony and 46 healthy horses. PROCEDURE: Basal plasma glucose (mg/dL) and insulin (mU/L) concentrations were determined from blood samples obtained between 8:00 AM and 9:00 AM. Minimal model results for 46 horses were compared by equivalence testing with proxies for screening SI and pancreatic beta-cell responsiveness in humans and with 2 new proxies for screening in horses (ie, reciprocal of the square root of insulin [RISQI] and modified insulin-to-glucose ratio [MIRG]). RESULTS: Best predictors of SI and AIRg were RISQI (r = 0.77) and MIRG (r = 0.75) as follows: SI = 7.93(RISQI) - 1.03 and AIRg = 70.1(MIRG) - 13.8, where RISQI equals plasma insulin concentration(-0.5) and MIRG equals [800 - 0.30(plasma insulin concentration 50)(2)]/(plasma glucose concentration - 30). Total predictive powers were 78% and 80% for RISQI and MIRG, respectively. Reference ranges and quintiles for a population of healthy horses were calculated nonparametrically. CONCLUSIONS AND CLINICAL RELEVANCE: Proxies for screening SI and pancreatic beta-cell responsiveness in horses from this study compared favorably with proxies used effectively for humans. Combined use of RISQI and MIRG will enable differentiation between compensated and uncompensated insulin resistance. The sample size of our study allowed for determination of sound reference range values and quintiles for healthy horses.

Effects of oral potassium supplementation on acid-base status and plasma ion concentrations of horses during endurance exercise.

OBJECTIVE: To compare effects of oral supplementation with an experimental potassium-free sodium-abundant electrolyte mixture (EM-K) with that of oral supplementation with commercial potassium-rich mixtures (EM+K) on acid-base status and plasma ion concentrations in horses during an 80-km endurance ride. ANIMALS: 46 healthy horses. PROCEDURE: Blood samples were collected before the ride; at 21-, 37-, 56-, and 80-km inspection points; and during recovery (ie, 30-minute period after the ride). Consumed electrolytes were recorded. Blood was analyzed for pH, PvCO2, and Hct, and plasma was analyzed for Na+, K+, Cl-, Ca2+, Mg2+, lactate, albumin, phosphate, and total protein concentrations. Plasma concentrations of H+ and HCO3-, the strong ion difference (SID), and osmolarity were calculated. RESULTS: 34 (17 EM-K and 17 EM+K treated) horses finished the ride. Potassium intake was 33 g less and Na+ intake was 36 g greater for EM-K-treated horses, compared with EM+K-treated horses. With increasing distance, plasma osmolarity; H+, Na+, K+, Mg2+, phosphate, lactate, total protein, and albumin concentrations; and PvCO2 and Hct were increased in all horses. Plasma HCO3-, Ca2+, and Cl- concentrations were decreased. Plasma H+ concentration was significantly lower in EM-K-treated horses, compared with EM+K-treated horses. Plasma K+ concentrations at the 80-km inspection point and during recovery were significantly less in EM-K-treated horses, compared with EM+K-treated horses. CONCLUSIONS AND CLINICAL RELEVANCE: Increases in plasma H+ and K+ concentrations in this endurance ride were moderate and unlikely to contribute to signs of muscle fatigue and hyperexcitability in horses.

Lipoic acid as an antioxidant in mature thoroughbred geldings: a preliminary study.

alpha-Lipoic acid (LA) has demonstrated antioxidant effects in humans and laboratory animals. The objective of this study was to determine whether the effects of LA are similar in horses. Five Thoroughbred geldings were supplemented with 10 mg/kg/d DL-alpha-lipoic acid in a molasses and sweet feed carrier and five received only the carrier as a placebo (CON). Blood samples were obtained at baseline (0 d), after 7 and 14 d of supplementation, and 48 h postsupplementation (16 d). Blood fractions of red and white blood cells (RBC and WBC, respectively) and plasma were analyzed for glutathione (GSH), glutathione peroxidase (GPx) and total plasma lipid hydroperoxides (LPO). An experienced veterinarian observed no adverse clinical effects. Plasma LPO baselines differed between groups (P = 0.002). When covariates were used, there was a decrease over time in the LA group (P = 0.015) and concentrations were lower in the LA group than in the CON group at 7 and 14 d (P = 0.022 and P = 0.0002, respectively). At baseline, GSH concentration was 69 +/- 7 in WBC and 115 +/- 13 mmol/mg protein in the RBC, with no differences resulting from either time or treatment. The GPx activity was 47 +/- 4 and 26 +/- 5 U/g protein at baseline WBC and RBC, respectively, with a lower concentration in the LA group's WBC at 7 (P = 0.019) and 14 d (P = 0.013). The results show that 10 mg/kg LA had no evident adverse effects, and moderately reduced the oxidative stress of horses allowed light activity. These findings encourage studying of LA in horses subjected to strenuous exercise.