Mature Horses Research Articles

28 A comparison among equine boots and legwraps on leg surface temperature during and after exercise

Boots and legwraps are commonly used to protect the lower limbs of the horse from trauma during exercise. However, previous research suggests such use can result in increased limb temperature that may be detrimental to underlying tendons. This study was designed to explore limb surface temperature during and after exercise when common leg protection was applied, hypothesizing that boots and wraps would increase temperature over the bare limb during exercise, yet show differences in the time required to return to baseline temperature. Six clinically-sound, mature horses (age 15 ± 2 yr; mean ± SEM) were used to test 6 boots or wraps: a neoprene boot, a perforated neoprene boot, an alternative neoprene boot, a cross country boot, a combination fleece and elastic wrap, and a polo wrap. The booted forelimb was selected randomly and the other forelimb served as a bare limb control in each test of a 6 × 6 Latin square design. An iButton hygrochron data logger was placed immediately palmar to the midpoint of the lateral splint bone on both the booted and bare limbs. The data logger recorded temperature and humidity every min during a 20-min standard exercise test and 180-min standing recovery during moderate ambient conditions (22.8 ± 0.9°C; humidity: 53.3 ± 4.8%). Data were analyzed using a mixed model design with repeated measures; significance was set at P < 0.05. The bare limb temperature was lowest (27.7 ± 1°C) at min 9 of the SET, then peaked and plateaued (33 ± 1°C; P < 0.05) during recovery. Conversely, treated limbs increased during exercise from 32 ± 1°C (min 0) to 36 ± 1°C at min 15, then plateaued during recovery. All treatment temperatures were greater than the bare limb (P < 0.0001) across all times. The polo wrap was hotter than the alternative neoprene sports medicine boot (P = 0.02), but neither was different from any other treatments. The humidity of the polo wrap was higher (P < 0.05) than all other treatments at 94%. All treatments failed to return to baseline temperature and humidity after 180 min (P = 0.01). The results support the hypothesis that convection cooling is impaired by boots and wraps during exercise. Given that skin temperature of the lower leg has been correlated as to underlying tendon surface temperature, this suggests that even in a moderate intensity exercise bout in moderate ambient conditions, a booted or wrapped limb may reach damaging temperatures. Contrary to our hypothesis, no boot or wrap nor the bare limb returned to baseline temperature in as much as 180 min of recovery, necessitating further research into the time required for passive cooling of the equine limb after exercise and verification of strategies to reduce cooling time.

Behavioral and cardiac responses in mature horses exposed to a novel object.

This study aimed to investigate whether breed, sex, and age affected temperament differently (more or less neophobic) in mature horses during a novel object test. The study included Jeju crossbred (n = 12, age = 9.42 ± 4.57 y), Thoroughbred (n = 15, age = 10.73 ± 3.09 y), and Warmblood horses (n = 12, age = 13.08 ± 3.55 y) with the females (n = 22, age = 11.36 ± 4.24 y) and geldings (n = 17, age = 10.65 ± 3.66 y). Jeju crossbreds (Jeju horse × Thoroughbred) are valuable considering their popular usage in Korea, but limited studies have explored temperament of Jeju crossbred horses. A trained experimenter touched the left side of the neck with a white plastic bag (novel object). The test ended when the horse stopped escape response and heart rate (HR) dropped to baseline. Behavioral score and escape duration were measured as behavioral variables. Multiple variables related to HR and heart rate variability (HRV) were measured to reflect emotional state. These included basal HR (BHR), maximum HR (MHR), delay to reach maximum heart rate (Time to MHR), standard deviation of beat-to-beat intervals (SDNN), root mean square of successive differences (RMSSD), and ratio of low to high frequency components of a continuous series of heartbeats (LF/HF). Statistics revealed that Thoroughbreds had significantly higher behavioral scores, and lower RMSSD than Jeju crossbreds (p < 0.05), suggesting greater excitement and fear to the novel object in Thoroughbreds. None of the behavioral or cardiac parameters exhibited sex differences (p < 0.05). Age was negatively correlated with SDNN and RMSSD (p < 0.05), indicating that older horses felt more anxiety to the novelty than younger horses. Thoroughbreds and females had distinct correlations between behavioral and HRV variables in comparison with other groups (p < 0.05), implying that escape duration might be a good indicator of stress, especially in these two groups. These results are expected to improve equine welfare, safety and utility, by providing insights into the temperament of particular horse groups, to better match reactivity levels with specific functions.

148 Analysis of purported insulin modulators on glycemic and insulinemic responses to various feeds in mature horses

Understanding the insulinemic and glycemic responses to common equine feeds and the efficacy of purported insulin modulators, such as aloe vera and grape seed extract, is valuable in designing appropriate equine diets. Using a repeated 5 × 5 Latin square design, dehydrated alfalfa (DA), beet pulp (BP), oats (O), steam-flaked corn (C), and steam-flaked corn with an insulin modulator consisting of combined aloe vera and grape seed extract (CIM) were tested for glycemic and insulinemic response in 10 mature horses aged 6 to 25 years with an average of 11.5 years. The various feeds were fed at the rate of 1.5 g/kg of body weight on an as-fed basis. The aloe vera and grape seed extract were fed at the rates of 150 mg/kg and 20 mg/kg of body weight respectively. Horses were maintained on a forage-only diet consisting of pasture grazing and hay, except once per week, when they were stalled and subjected to a glycemic response test. For testing, hay was removed the evening before, and a 12-h fasted blood sample was taken at time 0 before providing the assigned treatments. Horses were allowed 30 min to consume each treatment before any ORTS were removed. Additional blood samples were taken via jugular catheter at 30, 60, 90, 120, 150, and 180 min post-feeding. Blood glycemic and insulinemic responses were evaluated by maximum concentrations and time to peak response (minutes). Peak glucose and insulin were determined by Proc Mixed function in SAS 9.4 with the main effect of treatment and the random variables of horse and week. Results are presented as lsmeans ± SEM. Both DA and BP had lower peak glucose and insulin concentrations (P < 0.05) than the 3 high starch concentrate treatments (O, C, and CIM). The addition of insulin modulators to steam-flaked corn had no effect on the response for peak glucose [C: 128 ± 5 mg/dL, CIM: 133 ± 5 mg/dL, P = 0.39] or insulin [C: 54.5 ± 15.6 µIU/mL, CIM: 58.6 ± 15.6 µIU/mL; P = 0.78]. Likewise, the addition of the insulin modulators did not impact time to peak glucose concentrations [C: 93 ± 9 min, CIM: 108 ± 9 min, P = 0.23], nor the time to peak insulin concentrations [C: 81 ± 13 min, CIM: 87 ± 13 min, P = 0.73]. The results support that feeds with greater starch concentrations produce higher maximal blood glucose and insulin concentrations, but that, in this study, the addition of insulin modulators to steam-flaked corn did not result in a detectable change in the horses’ insulinemic and glycemic response.

68 Effect of a marine-sourced calcium on fecal pH in horses

Lithothamnium calcareum (LC) is a red, marine algae containing calcite in its cell walls. LC has been touted for its gastroprotective effects in horses attributed to the increased surface area of its honeycomb structure allowing greater acid-buffering capacity versus calcium carbonate. Effects on the equine hindgut have not been evaluated. This pilot study aimed to determine if LC (CalseaPowder, Nuwen, France) fed to horses at a rate of 4.6g/kg finished feed would alter fecal pH. Twelve mature horses (12.5 yrs ± 1.4, mean ± SEM, 6 mares, 6 geldings) in moderate exercise in the Ohio State ATI equine program participated in a randomized crossover study. Horses were maintained on 1.2-ha fields and brought into the barn for feeding and riding sessions. Horses transitioned onto the control feed over 2 wks (CON: SAFE ‘N EASY Performance, BUCKEYE Nutrition, Dalton, OH, 1.2% total Ca, 100% added was CaCO 3 ), formulated to meet or exceed NRC 2007 requirements for mature horses in light-moderate work. Horses were randomly assigned to receive either CON or CAL (CON + LC , 1.2% total Ca, added 74% CaCO 3 , 26% calcite) during the first test period then crossed over for the second. Horses acclimated to CON or CAL for 2 wks and were offered 0.5 kg feed per 100 kg bodyweight, divided into 2 feedings (0700 and 1600). Horses had access to mixed grass forage to supplement pasture, fresh water and salt ad libitum . At the end of the first 2-week trial period, horses were brought into stalls bedded with sawdust and observed. All fecal samples were collected immediately upon defecation (within 1–3 h post feeding) from the middle of the top of the manure pile to avoid bedding contamination and analyzed. Within 5 min, approximately 10 mL of fecal water was squeezed through cheesecloth from freshly defecated samples and stirred for 60s. Fecal water pH was then measured in duplicate with a calibrated pH meter. Diets were switched and the protocol repeated with 2 wk acclimation. Statistical analyses utilized R version 3.6.3, with significant differences declared when P ≤ 0.05. A linear mixed effect model was used with pH vs. diet and horse as a random effect. A Dunnett post-hoc comparison between the 2 diet groups was applied. There was no effect of period ( P = 0.66). There was an effect of diet ( P < 0.001). Horses consuming CAL had higher fecal water pH (6.87 ± 0.2; mean ± SD) compared with CON (6.69 ± 0.2). LC may be useful in supporting hindgut health in horses, especially when such changes in pH become clinically relevant (e.g., acidosis). Future work should examine the effects of different doses of LC on hindgut pH and its effects on the microbial population.

44 Relative preferences for cool season grass hay cultivars in adult horses

Tall fescue (TF) is productive in the Midwest and nutritionally similar to other cool season grasses. The potential for adverse effects has been documented in late gestation mares and growing horses, however effects on non-breeding mature horses are negligible. There are now more varieties of TF with novel endophytes designed to produce less ergovaline, the alkaloid to which toxicosis is attributed. The objective of this study was to evaluate relative preference for different monocultured hay by horses. In the fall of 2017, a field trial examining the performance of cool season grasses was established in Wooster OH. For the present study 7 were harvested: TF (v. KY 31; toxic endophyte), TF (v. Bronson; endophoyte free), TF (v. Texoma MaxQ; novel/non-toxic endophyte), Meadow Fescue (v. Preval), Festulolium (v. Spring Green), Perennial Ryegrass (v. Elgon), and Orchardgrass (v. Crown Royal) and a control was made by mixing all 7 cultivars together. First cutting chopped hay was created in 2019 and analyzed for nutrients and ergovaline concentrations. Ergovaline was below detectable limits in all samples, so 2020 hay was analyzed for nutrients and the presence of endophyte (via tiller staining) with only Bronson (3.3%) and Texoma Max Q (16.7%) having a low level of infection. Taste preferences were evaluated during summer 2019 and winter 2020 using a randomized complete block design with cultivar and bucket location as variables. Stalls had 8 buckets along a wall visible to pre-installed foaling cameras for recording. The same 4 mature horses at maintenance were used for the preference testing both years. Horses were fasted 30 min before entering the testing stalls and had access to the cultivars for 30 min. Weight consumed was recorded. Equine preference data for cultivar and bucket location were analyzed in JMP Pro 15. There was a significant difference (P < 0.001) in average consumption based on cultivar, however, standard deviations were high for all cultivars. In 2019 the cultivars in order of most to least preferred by the horses based on amount consumed (mean consumption ± standard deviation) were: 1) Perennial Ryegrass (2042 ± 243 g), 2) Orchardgrass (1258 ± 853), 3) Festulolium (1007 ± 900 g), 4) Control (771 ± 295 g), 5) TF Texoma MaxQ; non-toxic endophyte (451 ± 266 g), 6) TF Bronson; endophyte free (163 ± 86 g), 7) Meadow Fescue (118 ± 75 g), 8) TF KY31 (35 ± 98 g). Results were similar in 2020 with only the last 2 cultivars transposed in rank. Horses demonstrated a consistent preference for some species of cool season grasses in both 2019 and 2020, however the preference does not appear to be due to endophyte presence.

94 Feeding behavior and preference of horses fed rhizoma peanut hay

Rhizoma peanut has been used as an alternative legume hay for horses; however, feeding behavior and preference of horses fed this hay warrants investigation. This study aimed to evaluate the feeding behavior and preference of mature horses with maintenance requirements fed rhizoma peanut (RP, Arachis glabrata cv Florigraze) hay compared with alfalfa (ALF, Medicago sativa L. cv Legendary XHD) and bermudagrass (CB, Cynodon dactylon L. cv Coastal) hays. 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. After a 14-d diet adaptation, 24-h behavioral observations were conducted using scan sampling. Time spent feeding, at leisure, and at other activities were recorded every 10 min. Number of chews and bites were counted for 5 consecutive 1-min intervals. Preference tests were conducted on d 20 and 21 for a 2-h duration by simultaneously feeding 1 kg (as-fed basis) of each hay in separate trays twice a day (n = 4 observations/period). Horses’ first choice, hay consumption, and number of times each hay was smelled or tasted but left in favor of another hay were recorded. Hays were analyzed for chemical composition before the start of the study. Preference data were analyzed using descriptive statistics, while behavioral data and hay consumption were analyzed using repeated measures mixed model ANOVA. A hay x time of day effect was observed on feeding time ( P = 0.0393), whereas no hay x time effect was observed on time spent at leisure and other activities. When considering the total hours spent on each behavior, horses fed CB spent more time ( P = 0.0092) feeding compared with ALF (11.7 vs. 8.9 h/d) and less time ( P = 0.0037) at leisure compared with both legume hays. Hay type did not affect total time spent on other activities (1.8 ± 0.23 h/d; mean ± SE). Type of hay had no effect on bite rate (10.7 ± 2.2 bites/min), while horses fed CB had lower ( P = 0.0035) chew rate (71 chews/min) compared with RP and ALF (80 and 81 chews/min). Hay consumption was greatest ( P = 0.0425) with ALF (996 ± 17 g as-fed/test; mean ± SE), followed by RP (935 ± 30 g), and lowest with CB (792 ± 18 g). Digestible energy (Mcal/kg DM) in ALF was 2.58, while in RP was 2.53, and CB was 1.94. Crude protein concentrations in ALF, RP, and CB were 22, 16, and 13.6%, non-structural carbohydrates were 11.4, 10.1, and 7.8%, and neutral detergent fiber was 32.9, 40.4, 66.4%, respectively. Horses show preference for legumes, which might be associated with the nutrient profile of the hays. Rhizoma peanut provides time spent on feeding and chew rates similar to alfalfa.

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.

73 Impact of storage conditions on the viability of microbial inoculum for estimating in vitro digestibility in the horse

This study evaluated the effect of storage conditions of equine fecal material on the viability of derived microbial inoculum used for in vitro equine digestibility trials. Fecal material collected via rectal palpation from 3 mature quarter horse geldings was stored at 4 storage temperatures: 39°C for 15 min (control), 22°C for 6h, 3°C for 6h, and −18°C for 24h. Utilizing pooled microbial inoculum from stored fecal material, the in vitro digestion of 6 forage samples (NDF range 41 to 62%) consisting of 3 alfalfa and 3 mixed grass sample were digested in the Ankom Daisy II Incubator to evaluate interactions between storage condition of microbial inoculum and chemical composition of digested forage samples. After determination of dry matter digestibility (DMD), an Ankom2000 Fiber Analyzer was used to measure neutral detergent fiber digestibility (NDFD) and acid detergent fiber digestibility (ADFD). A mixed linear model was used to analyze differences in DMD, NDFD, and ADFD, with forage and treatment as class statements with date included as a random effect. No significant interaction of forage x treatment was observed ( P = 0.81 for DMD). Further analysis with a mixed linear model was used to evaluate the effect of forage quality parameters crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) as covariates for DMD, NDFD, and ADFD results. Significant differences were observed for DMD ( P < 0.0001 ), NDFD ( P < 0.01 ), and ADFD ( P < 0.001 ) between 39°C and 3°C, 39°C and −18°C, 22°C and 3°C, and 22°C and −18°C. No differences ( P > 0.05 ) were observed in DMD, NDFD, or ADFD between 39°C and 22°C or 3°C and −18°C. Covariance analysis found no difference ( P > 0.05 ) in slope between any of the 4 storage condition levels when CP, NDF, and ADF were modeled as covariates for DMD, NDFD, and ADFD. Results support that fecal material stored for up to 6 h in 22°C temperature conditions provide a viable alternative to fresh fecal material for the formation of microbial inoculum. When used to harvest inoculum for use in the Daisy II incubator fecal material that had been previously chilled or frozen did not result in DMD, NDFD or ADFD values of forage samples that were as high as when inoculum from the other 2 storage treatments were used. Further research is needed to determine the reason for the decline in DMD, NDFD, and ADFD when utilizing inoculum from chilled and frozen fecal samples.

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.

50 Intake, digestibility, and nitrogen balance in horses fed rhizoma peanut hay

Rhizoma peanut hay has the potential to meet horses’ crude protein requirements with reduced nitrogen excretion. This study aimed to evaluate nutrient intake, apparent digestibility, and nitrogen balance of rhizoma peanut (RP, Arachis glabrata cv Florigraze) hay compared with alfalfa (ALF, Medicago sativa L. cv Legendary XHD) and bermudagrass (CB, Cynodon dactylon L. cv Coastal) hays when fed to maintenance horses at 2% BW/d on a dry-matter (DM) basis. 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. Intake of nutrients is reported on a DM basis. Both legumes (ALF = 1.99% BW and RP = 1.97% BW) provided DM intake greater ( P < 0.05) than CB (1.82% BW) but were not different from one another. Digestible energy (DE) intake of ALF (29.9 Mcal/d) and RP (29.4 Mcal/d) were greater ( P < 0.05) than CB (20.8 Mcal/d). Crude protein (CP) intake was greater ( P < 0.05) for ALF (2,542 g/d), followed by RP (1,859 g/d), and then CB (1,477 g/d). Alfalfa provided the highest ( P < 0.05) calcium to phosphorus ratio of 6.3:1, while RP provided 4.4:1, and CB provided the lowest (1.7:1). Intake of all hays exceeded maintenance DE, CP, Ca, and P requirements. Apparent DM ( P < 0.05) and CP ( P < 0.05) digestibility were greater for ALF (69%, 84%), followed by RP (61%, 72%), and lowest for CB (46%, 64%). Neutral detergent fiber apparent digestibility did not differ ( P = 0.2228) among the 3 hays, while acid detergent fiber digestibility ( P = 0.0054) was lower for RP and similar for ALF and CB. Apparent digestibility of non-structural carbohydrate ( P < 0.05) was greater for ALF, followed by RP, and lowest for CB, while starch ( P < 0.05) digestibility was similar between the legumes and lowest for CB. Water intake for ALF was greater ( P < 0.05) than RP and CB. Greater urinary excretion ( P < 0.05) was observed for alfalfa, while water excreted through feces was greater ( P < 0.05) for CB, followed by RP, and lower for ALF. Urinary nitrogen (N) excretion was greater ( P < 0.05) for ALF, followed by RP, and lower for CB whereas fecal N excretion for both RP and CB were greater ( P < 0.05) than for ALF. Total N excreted was greater ( P < 0.05) for ALF (278 g/d), followed by RP (211 g/d), and lower for CB (179 g/d). Nitrogen retention was greater ( P < 0.05) for ALF when represented as g/d, but similar to RP when presented as percent of N intake. Results indicate that rhizoma peanut hay is a suitable legume for horses by meeting DE and CP requirements with significantly less N excretion than alfalfa.

21 Mucosal IgA displays an unclear pattern throughout the day in horses

Mucosal immunoglobulin (Ig) A is a useful indicator of immunity, a sensitive biomarker for stress and a desirable target for more effective vaccine development. In human medicine, treating diseases at specific times of the day, known as chronotherapy, is rapidly becoming an accepted practice. Equine research suggests protection obtained from vaccines may be enhanced with timed delivery. Reports of diurnality of both fecal and salivary IgA exist in other species, but this has not been confirmed in horses. The aim of this preliminary study was to investigate IgA for 24-h rhythmicity. Four mature Quarter horse geldings (mean ± SEM, 600 ± 27 kg; 10.3 ± 4.0 y) were housed according to their normal daily routine under natural summer conditions; outside in individual dry lots for 12 h and stalled inside overnight. Sunrise and sunset were at 0600 and 2000 h, respectively. Horses were fed the same quantities of concentrate and bermudagrass hay twice daily. Venous blood, fecal and saliva samples were collected at 6-h intervals (0600, 1200, 1800, 2400 h) for 4 consecutive days using only headlamps to collect samples after sunset. Cortisol and IgA concentrations were determined by ELISA. Data were log-transformed, blocked by day, and compared using mixed model ANOVA with time as a repeated variable. Serum cortisol displayed an expected cyclical pattern with the greatest concentrations observed at 0600 h ( P < 0.01; 54.7 ± 4.6 ng/mL) followed by a decrease throughout the day, reaching a nadir at 2400 h ( P < 0.05; 25.5 ± 2.4 ng/mL). Fecal IgA lacked a clear pattern and was variable within and between horses. Within a 24-h period, the lowest and highest concentrations of fecal IgA were 394.5 ± 105.5 to 2246.7 ± 1576.9 ng/mL. Considering the gut transit time for this molecule, more consecutive days of sampling may be required to distinguish rhythmicity. Salivary IgA concentration was highest at 2400 h (809.4 ± 137.5 ug/mL) and lowest at 1200 h (590.2 ± 138.9 ug/mL). Salivary IgA was higher at 2400 h compared with 1200 h ( P = 0.01) and 1800 h ( P = 0.06). Salivary IgA was also higher at 0600 h compared with 1200 h ( P = 0.05). Serum IgA did not display a diurnal pattern but did decrease ( P < 0.0001) over the 4 d of sampling. These data suggest daily fluctuations may be normal and should be accounted for by establishing a solid baseline and/or using a covariate during analysis. More research is needed to elucidate 24-h rhythmicity of IgA and determine how variations in in this molecule may be applied to chronotherapy and vaccine delivery in horses.

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.

45 An assessment of olfactory influence on equine feeding preferences

Few data are available relating the impact of olfactory stimuli to the feeding preferences of horses. Anise has been demonstrated to increase palatability of equine feed products but has not been evaluated for its olfactory impact. The objective of this study was to evaluate the impact of an olfactory stimuli, anise, on feed preference and behaviors. Eight mature stock-type horses group-housed with ad libitum access to pasture (cool season grass/legume mix), water and salt were used in this paired preference test across 6 d of testing. Anise oil was compared with a control (corn oil) in a crossover design to determine the effects of olfactory stimulation via a known palatant on food preference in horses. Horses were loaded into identical feeding stocks with a feeding bunk containing 2 separated sides. The false bottom pans were constructed with 24 equally spaced openings (0.20 cm) and one (0.20 cm) central opening to allow odor permeation. Feeding pans were loaded with 227 g of crimped oats and situated on top of plates containing gauze squares containing 0.375 mL of either control (corn) oil or anise oil. Horses were considered “finished” when > 75% of one diet was consumed. Preference was described as a higher number of sniffing episodes or larger consumption. Variables of interest included first diet sniffed and consumed, first action, aversive behaviors, and excessive salivation. Aversive behaviors were described as rejection of the product accompanied by a combination of behaviors including extreme head extension, retracted lips, gaping mouth, full tongue protrusion, and backward rotation of the ears. Excessive salivation was described as saliva coating the outer lips or dripping from the corners of the mouth while chewing. Each trial was video-recorded, and behavior data were analyzed using a chi-squared analysis with SAS version 9.4. Data for total consumption were analyzed using a Kruskal-Wallis test with P ≤ 0.05 set as significant for all data. Horses sniffed anise first more frequently ( P = 0.02) and similarly consumed anise first more frequently than control ( P = 0.04). Accordingly, anise was finished first more often than control ( P < 0.01). Interestingly, horses were more likely to sniff the offered diets as compared with consumption when first actions were compared ( P < 0.01). Not surprisingly, anise was consumed in twice the quantity as compared with control (145.5g and 71.99g, respectively; P < 0.01). These novel data represent a highly significant relationship between olfactory stimuli and feeding preferences in horses. More work is needed to determine threshold and sensitivity to novel aromas.

Osteochondral fragmentation of the cervical articular process joints; prevalence in horses undergoing CT for investigation of cervical dysfunction.

Computed tomography (CT) imaging of the cervical spine of mature horses under general anaesthesia is becoming increasingly accessible. Osteochondral fragmentation (OF) of the cervical articular process joints (APJs) has been identified on CT imaging; the prevalence, clinical features and significance of this finding are currently unknown. To describe the prevalence and clinical features of a population of horses with CT evidence of OF within the cervical APJs. Retrospective, descriptive case series. The study population consisted of all horses undergoing CT imaging of the entire cervical spine between April 2016 and December 2019 at Liphook Equine Hospital for investigation of clinical signs localised to this region. CT scans were examined for evidence of OF within the APJs and additional case features were recorded. A total of 55 horses underwent CT imaging during the study period. OF was identified in 13 horses, giving a CT prevalence of 24% in the study population. OF occurred in a range of ages, breeds and uses of equine, presenting with varied clinical signs. It was identified at every level of the neck. Additional cervical pathology was identified in 11/13 OF cases. Retrospective analysis of a clinical caseload with a lack of clinically normal controls or post mortem confirmation of imaging findings. Low case numbers. OF within the cervical APJs is recognised on CT imaging in horses with cervical dysfunction. Fragments are commonly identified in combination with additional cervical pathology. Further investigation is required to confirm their clinical significance and develop potential treatment options where necessary.

Changes in metabolic and physiological biomarkers in Mangalarga Marchador horses with induced obesity

This study aimed to characterize and correlate physiological and metabolic changes in horses fed a hypercaloric diet (HD). Nine mature horses with a mean initial body condition score of 2.9 ± 1 (scale, 1–9) were fed a high-calorie diet for 5 months. Fasting blood samples were collected before the study and biweekly for the duration of the project to determine the concentrations of cholesterol (CHOL), very low (VLDL), low (LDL) and high-density (HDL) lipoproteins, triglycerides, non-esterified fatty acids, and fructosamine. A low-dose oral glucose tolerance test (LGTT) was conducted before, 75 and 150 days after HD introduction. Mean arterial blood pressure was measured monthly. Following HD introduction, CHOL, LDL, HDL, and fructosamine blood concentrations increased (P < 0.001). These four variables were also positively and significantly correlated with the blood insulin response to LGTT. These findings confirm the occurrence of hypercholesterolemia concomitantly with insulin dysregulation development in horses exposed to HD.

Horses in the Late Tagar Economy: Kosogol I Settlement Materials (Krasnoyarsk Region)

The archaeological studies developed two concepts concerning the character of Tagar economy: semi-nomadic and sedentary. They are based on different approaches to burial assemblage materials, accidental findings, and general opinion on stock-raising economy in Eurasian forest-steppes and steppes. The zooarchaeological profile of Tagar settlements can resolve this argument. A high share of horse bones may mean a semi-nomadic or nomadic lifestyle, while low share of equine remains can be a sign of a sedentary economy. The research featured Tagar settlements in the forest-steppe areas of the interfluve area between the Kiya and the Chulym. The paper describes the zooarchaeological collection of the archeological site of Kоsоgol I, the largest Early Iron Age settlement in the area. The collection includes 6,634 samples, of which 687 belong to horses. The authors believe that cattle breeding was the main branch of the Tagar economy. Horses were the third most important group. However, horses were not meat animals, as bones of young horses were quite rare among the kitchen waste. The Tagars killed mature or old work horses (older than 12–13), which could not work anymore. Hunting was a secondary branch of their economy: they hunted does, as well as water and moor fowl near the settlement. The results of Kosogol I zooarchaeological assemblage study proved the theory about the sedentary cattle breeding of the early Tagar people.

Effect of bioactive proteins on gait kinematics and systemic inflammatory markers in mature horses.

Twenty-seven mature Quarter horses were used in a randomized design to determine the effects of bioactive protein supplementation on gait kinematics and systemic inflammatory markers in a 34-d trial. Treatments consisted of oral doses of 230 g/d of pelleted supplements containing 0 g (CON; n = 9), 40 g of bioactive protein (40BP; n = 9; LIFELINE, APC, LLC, Ankeny, IA), and 80 g of bioactive protein (80BP; n = 9) daily. Horses were fed a commercial concentrate at 0.5% BW (as-fed) and received ad libitum coastal bermudagrass (Cynodon dactylon) hay daily. On day 33, horses consistent in exercise (CON, n = 6; 40BP, n = 8; 80BP, n = 7) participated in a trailering and riding challenge. Kinematic gait analysis was performed on day 0 for use as a covariate, and on day 14, 28, and 34 to allow for the determination of potential time and dosage effects. Video footage was collected and analyzed using gait analysis software (EquineTec, Monroe, GA) for the determination of stride length (SL) and range of motion (ROM). Blood was collected via jugular venipuncture on days 0, 14, 28, and 34 for determination of systemic expression of tumor necrosis factor (TNF)-α and IL-1β. Data were analyzed using PROC MIXED of SAS. A trend towards treatment × time interaction was observed in ROM of the knee at the walk (P = 0.10), due to the increasing ROM for 40BP and 80BP as time increased and decreasing ROM for CON. A treatment × time interaction was observed (P < 0.01) for hock ROM at a walk resulting from CON and 80BP decreasing from day 14 to 28 with 40BP increasing, while from day 28 to 34 ROM at a walk decreased for 40BP and increased for 80BP. The main effect of treatment on hock ROM at the walk was quadratic (P < 0.01) and characterized by higher ROM values for 40BP compared to CON or 80BP. Dietary treatment lengthened (P = 0.04) SL of the hind limb at the walk for 40BP and 80BP compared to CON on both days 14 and 28. A significant treatment × time interaction was observed in the expression of IL-1β (P < 0.01) and can be explained by lower concentrations of IL-1β for 80BP on day 34 compared to the other treatments, with 40BP being intermediate and CON being the highest. Increased articular ROM with decreased expression of IL-1β may indicate potential anti-inflammatory effects of 80 g/d of bioactive proteins.

Effect of hay type on cecal and fecal microbiome and fermentation parameters in horses.

The effect of hay type on the microbiome of the equine gastrointestinal tract is relatively unexplored. Our objective was to characterize the cecal and fecal microbiome of mature horses consuming alfalfa or Smooth Bromegrass (brome) hay. Six cecally cannulated horses were used in a split-plot design run as a crossover in two periods. The whole plot treatment was ad libitum access to brome or alfalfa hay fed over two 21-d acclimation periods with subplots of sampling location (cecum and rectum) and sampling hour. Each acclimation period was followed by a 24-h collection period where cecal and fecal samples were collected every 3 h for analysis of pH and volatile fatty acids (VFA). Fecal and cecal samples were pooled and sent to a commercial lab (MR DNA, Shallowater, TX) for the amplification of the V4 region of the 16S rRNA gene and sequenced using Illumina HiSeq. The main effects of hay on VFA, pH, and taxonomic abundances were analyzed using the MIXED procedure of SAS 9.4 with fixed effects of hay, hour, location, period, and all possible interactions and random effect of horse. Alpha and beta diversities were analyzed using the R Dame package. Horses fed alfalfa had greater fecal than cecal pH (P ≤ 0.05), whereas horses fed brome had greater cecal than fecal pH (P ≤ 0.05). Regardless of hay type, total VFA concentrations were greater (P ≤ 0.05) in the cecum than in feces, and alfalfa resulted in greater (P ≤ 0.05) VFA concentrations than brome in both sampling locations. Alpha diversity was greater (P ≤ 0.05) in fecal compared with cecal samples. Microbial community structure within each sampling location and hay type differed from one another (P ≤ 0.05). Bacteroidetes were greater (P ≤ 0.05) in the cecum compared with the rectum, regardless of hay type. Firmicutes and Firmicutes:Bacteroidetes were greater (P ≤ 0.05) in the feces compared with cecal samples of alfalfa-fed horses. In all, fermentation parameters and bacterial abundances were impacted by hay type and sampling location in the hindgut.

Clinical implications of using adrenocorticotropic hormone diagnostic cutoffs or reference intervals to diagnose pituitary pars intermedia dysfunction in mature horses.

BackgroundDiagnosis of pituitary pars intermedia dysfunction (PPID) is problematic because of large variations in ACTH concentrations.Hypothesis/ObjectivesCompare the test characteristics of baseline and post–thyrotropin‐releasing hormone (TRH) stimulation plasma ACTH concentrations in horses using diagnostic cutoff values (DCOVs) and reference intervals (RIs) and determine the clinical consequences of using each method.AnimalsOne hundred six mature horses: 72 control cases and 34 PPID cases.MethodsProspective case‐controlled study. Horses underwent monthly TRH stimulation tests. Diagnostic cutoff values were determined monthly by receiver operating characteristic curves using the Youden index. Reference intervals were determined monthly by a robust method. For each case age, sex and body condition score (BCS) were recorded.ResultsBaseline ACTH concentrations varied by month (P < .001) with significant “month × age” (P = .003), “month × sex” (P = .003), and “month × BCS” (P = .007) effects. Baseline ACTH concentrations were accurate to diagnose PPID (0.91 ± 0.06) with DCOVs increasing the test sensitivity (0.61 ± 0.21 to 0.87 ± 0.05, P = .002) and RI increasing test specificity (0.85 ± 0.12 to 0.98 ± 0.01, P = .01). Thyrotropin‐releasing hormone stimulation improved test accuracy (0.91 ± 0.06 to 0.97 ± 0.03, P = .004).Conclusions and Clinical ImportanceACTH concentrations follow a circannual rhythm and vary with physiological factors. As using DCOVs increases the ability to detect mild cases and using RI decreases the risk of unnecessary treatments, ACTH concentrations should be interpreted within a specific clinical context. The TRH stimulation test improves the diagnosis of PPID.

66 Intra-articular lipopolysaccharide challenge has no long-term effects on inflammation and cartilage metabolism

Abstract Intra-articular lipopolysaccharide is a common model to induce acute synovitis to investigate the effects of various therapeutic agents or nutraceuticals. The long-term effects of intra-articular lipopolysaccharide use in skeletally mature and immature horses has yet to be investigated; therefore, the objective of this study was to describe long-term effects of single-administration of intra-articular lipopolysaccharide on joint inflammation and cartilage metabolism. To test this objective, both radial carpal joints from 5 stock-type horses never exposed to lipopolysaccharide (CON; n = 10) were compared to radial carpal joints from 17 similar stock-type horses previously exposed to intra-articular lipopolysaccharide (INFL; n = 34). Joints within INFL were further categorized as the joint which received lipopolysaccharide (LPS; n = 17) and contralateral control which received iso-volumetric lactated Ringer’s solution (CONTRA; n = 17). A single synovial fluid sample from each joint was analyzed for prostaglandin E2 (PGE2), collagenase cleavage neopeptide (C2C), carboxypeptide of type II collagen (CPII), and chondroitin sulfate 846 (CS846). All data were analyzed using PROC MIXED of SAS with main effects of treatment (CON, INFL) and joint (CON, LPS, CONTRA). Time post-administration (1.5, 2, 6 yr) and age-at-administration (1, 3, 5, 7 yr) were included in the model within INFL joints (LPS, CONTRA). There was no influence of treatment on any biomarker (P &amp;gt; 0.40). Similarly, inflammation and cartilage metabolism were not different between CON, CONTRA, and LPS joints (P &amp;gt; 0.50). Within INFL, there was no influence of joint, age, or time post-administration for PGE2, CPII, or CS846 (P &amp;gt; 0.10). A joint x time interaction was observed for catabolic C2C (P &amp;lt; 0.01); however, where LPS was less than CONTRA 2 yr post-lipopolysaccharide administration and similarly when lipopolysaccharide was administered at 5 and 7 yr of age (P &amp;lt; 0.01). These data indicate no long-term negative effects for the use of intra-articular lipopolysaccharide as an acute inflammatory model in skeletally mature and immature horses.