Phosphorus excretion by mares post-lactation.

Across the equine literature, estimates of true P digestibility range from -23% to 79%. This large range cannot be explained by differences in P intake or phytate-P intake alone. However, differences in endogenous P secretion into the GI tract may explain the variation. In horses, excess absorbed P is not excreted in the urine but is re-secreted into the GI tract, increasing faecal P and leading to estimates of low P digestibility. Thus, accurate estimates of P digestibility can only be obtained if absorbed P is retained in the horse. The objective of this study was to examine P digestibility in post-lactational mares and control mares that were fed similar amounts of P. It was hypothesized that post-lactational mares would have greater P retention and higher apparent P digestibility than control mares. Prior to the study, four lactating and four non-lactating mares were fed a diet that provided 100% of the control mares' P requirement, but only 55% of the lactating mares' P requirement. During the study, both groups were fed P at the rate recommended for non-lactating mares. Post-lactational mares did not retain more P than control mares but tended to excrete more P than control mares (p = .082), presumably due to differences in endogenous P secretion into the GI tract. Metabolic changes occurring during mammary gland involution may have contributed to the increase in P excretion. However, faecal P excretion exceeded P intake in both groups (p = .08) and both groups lost weight during the study. Tissue mobilization during weight loss may have influenced P secretion into the GI tract.

Improved methods for mare milk analysis: Extraction and quantification of mare milk carbohydrates and assessment of FTIR-based macronutrient quantification.

Accurately determining the macronutrient profile of mare milk is a precursor to studying how milk composition affects foals' growth and development. This study optimized and validated an extraction and quantification method for mare milk oligosaccharides, which make up a portion of the carbohydrate fraction of mare milk. Mare milk was extracted with chloroform and methanol, and oligosaccharides were selectively isolated from the carbohydrate fraction using porous-graphitized carbon solid-phase-extraction (SPE). Good recovery rates for milk oligosaccharides (between 70 and 100%) were achieved with the optimized method. This study also compared the use of Fourier-Transform infrared (FTIR) spectroscopy versus wet chemistry quantification methods for protein, fat, and lactose. The FTIR method produced statistically equivalent protein contents to the wet chemistry method, along with substantial savings in both analyst time and consumable consumption. FTIR analysis slightly underestimated the fat content of mare milk relative to the official wet chemistry method, with the difference between the methods increasing at higher fat contents. FTIR also overestimated the lactose content of mare milk and appeared to generate "lactose" values that included the milk oligosaccharides and thus represented the total carbohydrate (lactose and milk oligosaccharides) content of mare milk.

Technical Note: A method for determination of titanium dioxide concentration in fecal samples.

Titanium dioxide has been used as a marker for determining diet digestibility indirectly, but some authors have expressed difficulty in measuring TiO2 concentrations in fecal material. We developed an accurate and precise method to determine TiO2 concentrations in equine feces. The method includes dry-ashing samples, digestion with (NH4)2SO4 in concentrated sulfuric acid, followed by the addition of H2O2 to produce a yellow to orange color that can be read spectrophotometrically. Accuracy was tested by spike recovery, and precision was tested by examining the coefficient of variation (CV) between duplicates of 449 individual samples. The method described here was compared with a previously published method by examining CV between duplicates of samples analyzed using both methods and comparing them using a paired t-test. Titanium dioxide spike recovery averaged 106%, and the CV between duplicates averaged 4.0%, with 79% of sample pairs having a CV of <5%. When compared with a previously published method, the method described here had a lower CV between duplicates (P < 0.0001). The method described here provides an accurate and precise quantitative analytical procedure for TiO2 in equine fecal samples.

Titanium dioxide is a marker fed to animals to help determine diet digestibility indirectly by measuring the concentration of TiO2 in fecal samples. This paper describes an accurate and precise method to analyze TiO2 in equine feces. The precision of this method is demonstrated by the low variation between sample duplicates. This method requires fewer sample replicates for analysis, leading to less labor, expense, and waste in the laboratory.

Ethanol-Soluble Carbohydrates of Cool-Season Grasses: Prediction of Concentration by Near-Infrared Reflectance Spectroscopy (NIRS) and Evaluation of Effects of Cultivar and Management.

Ethanol-soluble carbohydrates (ESCs) of cool-season grasses include mono- and disaccharides and sometimes short-chain fructans, which may exacerbate the risk of pasture-associated laminitis. A calibration for prediction of ESC concentrations by near-infrared reflectance spectroscopy (NIRS) was developed from 323 samples of four cool-season grass species (orchardgrass, Kentucky bluegrass, tall fescue, and perennial ryegrass) across 10 cultivars collected in central Kentucky in the morning and afternoon over two growing seasons. The calibration, which had accuracy above 95%, was used to predict ESC concentrations of 1,532 samples from the second growing season. ESC concentrations increased in the afternoon compared to the morning across all cultivars. In the majority of samples, ESC concentrations were not affected by nitrogen application to plots. Use of NIRS has the potential to evaluate management and cultivar effects on ESC concentrations in cool-season grass pastures.

Water-Soluble Carbohydrates of Cool-Season Grasses: Prediction of Concentrations by Near-Infrared Reflectance Spectroscopy and Evaluation of Effects of Genetics, Management, and Environment.

An excess of water-soluble carbohydrates (WSCs) can present a risk for horses prone to pasture-associated laminitis or some other metabolic conditions. Determining WSC concentrations in commonly grazed cool-season grasses, at different times of day and under different fertilization treatments, can help optimize grazing times and management strategies. The goals of this study were to develop a near-infrared reflectance spectroscopy (NIRS) calibration curve for WSC and to apply it to four cool-season grass species. Tall fescue, Kentucky bluegrass, orchardgrass, and perennial ryegrass (10 cultivars) were sampled every two to four weeks from plots with or without added nitrogen, in the morning and afternoon. WSCs were quantified colorimetrically for a sample subset, and these values were used to develop an NIRS calibration predicting WSC concentrations with 90% accuracy. An interaction of species, nitrogen treatment, time of day, and harvest date influenced WSC concentrations in 10 harvests (P = .040). A modest positive relationship was observed between photosynthetically active radiation and WSC concentration when morning and afternoon samples were included (r = 0.503; P = .024). On nine harvest dates, perennial ryegrass or tall fescue were highest in WSC. High-WSC cultivars included "Aberzest" and "Calibra" perennial ryegrass, "Ginger" Kentucky bluegrass, and "Bronson" and "Cajun II" tall fescue. Water-soluble carbohydrates did not exceed 150 g/kg freeze-dried weight, possibly due to assay method, sampling times, or defoliation. The results suggest that minimizing WSC intake for horses may be possible by cultivar choice, grazing time, or mowing frequency.

Relationships Between Measurements of Body Fat in Thoroughbred Horses.

Equine obesity is increasing in prevalence, and weight loss diets are frequently recommended for these horses. However, there are also management situations in which horses are deemed to be too thin. To monitor the efficacy of weight change programs, estimates of body fat are often made. There are several systems available to estimate body fat, and there are benefits and challenges to using each method. The objective of this study was to compare four different methods of estimating body fat in Thoroughbred horses. In 14 mature Thoroughbred horses, relationships among body condition score (BCS), morphometric measurements, ultrasonic measures of subcutaneous fat depots, and estimation of total body fat (BFD) via measurement of total body water through deuterium oxide dilution were evaluated. Body condition scores ranged from 4.5 to 6.5 on a 9-point scale. Body condition score, heart girth-to-body weight ratio, and BFD were all positively correlated with each other (P < .05). Subcutaneous fat depth at the tailhead tended to be positively related to BFD when only horses with BCS ≥ 5 were included (P = .0680). These data suggest that BCS remains a simple means of monitoring adiposity in mature horses in moderate condition. Tailhead fat depots may become useful for monitoring changes in body fat in Thoroughbreds with a BCS above 5, although more work with animals of higher adiposity is required and at different times of year.

Seasonal and Diurnal Variation in Water-Soluble Carbohydrate Concentrations of Repeatedly Defoliated Red and White Clovers in Central Kentucky.

Nonstructural carbohydrates of pasture plants, comprising water-soluble carbohydrates (WSCs) and starch, may contribute to excessive consumption of rapidly fermentable carbohydrates by grazing horses. Seasonal and diurnal variation in WSCs were studied in red (Trifolium pratense L.) and white clovers (Trifolium repens L.) subjected to a typical management regime of rotationally grazed horse pastures. Two red and two white clover cultivars from monoculture plots were harvested after 4 weeks of growth from April to October of 2015, in the morning and afternoon of each harvest date. Water-soluble carbohydrates were quantified for each harvest, and starch was quantified for two harvests. Mean monthly WSC concentrations ranged from 80 to 99 mg/g (freeze-dried weight basis), whereas mean starch concentrations were 31 and 40 mg/g. In September, white clover had 14% more WSCs than red clover (P < .0001). Water-soluble carbohydrate concentrations were 10% higher in the afternoon than in the morning (P < .0001). Starch concentrations were 290% higher in the afternoon than in the morning (P < .0001), and nonstructural carbohydrate concentrations in the afternoon averaged 150 mg/g. Further studies are needed to determine whether the mixed grass-legume pastures of central Kentucky accumulate enough nonstructural carbohydrates to present risk factors for equine metabolic or digestive dysfunction.

Effect of body condition, body weight and adiposity on inflammatory cytokine responses in old horses.

Advanced age is associated with a low-grade, systemic inflammatory response characterized by increased inflammatory cytokine production both in vitro and in vivo, termed inflamm-aging. It is also known that increased white adipose tissue, associated with obesity, leads to increased production of inflammatory cytokines. To date, it is unknown whether increased adiposity contributes to the age-related increased inflammatory status. Here we show that peripheral blood mononuclear cells (PBMC) from old horses compared to young horses have increased inflammatory cytokine production; moreover, fat old horses compared to thin old horses have even greater frequencies of lymphocytes and monocytes producing inflammatory cytokines. Therefore, we proposed that decreasing adiposity in old horses would reduce age-associated increases of inflammatory cytokines both in vitro and in vivo, and increasing adiposity in old horses would increase these measurements. To test this hypothesis further, eight old obese horses (20-28 year) were assigned to two consecutive treatments, dietary restriction (DR) during weeks 1-12 and increased dietary intake (DI) during weeks 13-30. Body weight, body condition score (BCS) and percent body fat were measured weekly. PBMC were stimulated in vitro and interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) production was measured by intracellular staining. Levels of nascent IFNgamma and TNFalpha mRNA expression were examined by RT-PCR. Serum concentrations of TNFalpha protein were also measured weekly. Reducing body weight and fat in old horses significantly reduced the percent of IFNgamma and TNFalpha positive lymphocytes and monocytes, and serum levels of TNFalpha protein. Further, when weight and fat increased in these old horses there was a significant increase in inflammatory cytokine production. Regression analysis also revealed significant relationships. These findings demonstrate that age-related obesity potentially plays a role in the dysregulation of inflammatory cytokine production by the immune system with age or inflamm-aging in the horse.

The effect of diagnosis-specific computerized discharge instructions on 72-hour return visits to the pediatric emergency department.

OBJECTIVE: The number of patients returning to the pediatric emergency department (PED) within 72 hours of discharge is frequently cited as a benchmark for quality patient care. The purpose of this study was to determine whether the introduction of diagnosis-specific computer-generated discharge instructions would decrease the number of medically unnecessary return visits to the PED. METHODS: A retrospective chart review of patients who returned to the PED within 72 hours of discharge was performed. Charts were reviewed from 2 comparable periods: September 2004 to February 2005, when handwritten discharge instructions were issued to each patient, and September 2005 to February 2006, when each patient received computer-generated diagnosis-specific discharge instructions. The patient's age, primary care provider, insurance status, chief complaint, vital signs, history, physical examination, plan of care, and diagnosis at each visit were recorded. Cases were excluded if the patient left against medical advice or without being seen, was admitted to the hospital on the first visit, or had incomplete or missing records. The medical necessity of the return visit was rated as "yes," "no," or "indeterminate" based on review of the visit noting reason for return, history and physical examination, diagnosis, and interventions or changes in the initial care plan. RESULTS: Of all return visits to the PED within 72 hours of discharge, 13% were deemed unnecessary for patients receiving handwritten instructions compared with 15% for patients receiving computer-generated instructions (P = 0.5, not significant). For each additional year of age, the return visit was 1.07 times as likely to be medically appropriate (95% confidence interval, 1.03-1.12; P = 0.002). Patients who returned to the PED more than once were 2.69 times more likely to have a medically appropriate visit as were those with only 1 return visit (95% confidence interval, 0.95-7.58; P = 0.062). CONCLUSIONS: Computer-generated diagnosis-specific discharge instructions do not decrease the number of medically unnecessary repeat visits to the PED.

Effects of Harvest Date, Sampling Time, and Cultivar on Total Phenolic Concentrations, Water-Soluble Carbohydrate Concentrations, and Phenolic Profiles of Selected Cool-Season Grasses in Central Kentucky.

Grasses are a source of nutrients for grazing horses. However, water-soluble carbohydrates (WSCs) of grasses have been implicated in some equine health issues. Grasses also contain phenolic compounds, whose sensory and antimicrobial properties may affect forage intake by horses and horse health. The goals of this study were to assess factors affecting phenolic and WSC concentrations in selected cultivars of cool-season grasses and profile the phenolic compounds. Total phenolics and WSC were quantified in "Linn" and "Calibra" perennial ryegrass (PRG), "Cajun II" tall fescue (Cajun TF), "Persist" orchardgrass (OG), and "Ginger" Kentucky bluegrass (BG), collected in the morning and afternoon of late April, early May, and late May. WSCs were higher in the afternoon (P < .0001), and afternoon concentrations differed among cultivars (P = .011) and decreased by late May (P < .0001). Total phenolics, initially highest in Calibra PRG and Persist OG, decreased or remained constant from late April to late May, except in Persist OG (P < .0001). Total phenolics decreased in the afternoon in Persist OG and Calibra PRG (P = .015). High-performance liquid chromatography revealed similar phenolic profiles in Calibra PRG, Linn PRG, and Cajun TF. These differed from the profiles of Ginger BG and Persist OG. Major peaks were characterized by liquid chromatography coupled to mass spectrometry. The cultivars had a diversity of phenolic compounds possibly worth exploring for properties that may interact with those of WSC to impact equine health.

Effect of Starch Source in Pelleted Concentrates on Fecal Bacteria in Prepartum and Postpartum Mares.

Dietary starch source has been shown to affect fecal bacterial communities of horses fed minimally processed cereal grains. However, processing may increase foregut starch digestibility, reducing effects of starch source on fecal bacterial communities. This study aimed to determine the effect of starch source in pelleted concentrates on fecal Lactobacillus spp., amylolytic bacteria, and cellulolytic bacteria in broodmares mares, during the prepartum and postpartum period. Thoroughbred mares (n = 18) were paired by last breeding date then randomly assigned to either an oat-based or a corn and wheat middlings-based pelleted concentrate fed with forage. Mares were fed their assigned concentrates beginning on 310 days of gestation, and fecal samples were collected at 324 days of gestation, before parturition, 1 day, 14 days, and 28 days postpartum. Fecal samples were enumerated by serial dilution and inoculation into selective, enriched media for Lactobacillus spp., amylolytic bacteria, and cellulolytic bacteria. Data were log transformed then analyzed using a mixed model ANOVA with repeated measures (SAS 9.3) to test the main effects of treatment, time of sample, and treatment by time interaction. Starch source did not affect enumerated bacterial communities (P > .05); thus, pelleting concentrates may alter some of the effects of starch sources on the hindgut microbiota. Sample date did not affect amylolytic bacteria (P > .05); however, lactobacilli and cellulolytic bacteria decreased 1 day postpartum (P < .05). Although we did not observe an effect of starch source on fecal bacteria in mares, parturition did appear to alter the hindgut microbiota.

Effect of maternal diet on select fecal bacteria of foals.

Adult horses depend on the microbial community in the hindgut to digest fiber and produce short-chain fatty acids that are use for energy. Colonization of the foal gastrointestinal tract is essential to develop this symbiosis. However, factors affecting colonization are not well understood. The objectives of this study were to evaluate the age-related changes and effects of maternal diet on select fecal bacterial groups in foals from 1 to 28 d of age. Thoroughbred foals (n = 18) were from dams fed forage and one of two concentrates: an oat-based (OB) or corn and wheat middlings-based (CWB) pelleted concentrate. The mares had access to assigned concentrates, along with a mixed hay and cool-season grass pasture, 28 d before and 28 d after parturition. Fecal samples were collected from foals at 1 d (14 to 36 h), 4, 14, and 28 d after birth. Fecal samples were serially diluted with phosphate-buffered saline before inoculation of enriched, selective media to enumerate Lactobacillus spp., amylolytic bacteria, and cellulolytic bacteria. Enumeration data were log-transformed then analyzed with mixed model analysis of variance with repeated measures (SAS 9.3) to test the main effects of maternal diet (OB or CWB), time of sample, and interaction between maternal diet and time. Cellulolytic bacteria first appeared in foal feces between 4 and 14 d of age and increased with age (P < 0.05). Amylolytic bacteria and lactobacilli were abundant at 1 d and then increased with age (P < 0.05). There was an interaction between maternal diet and time for Lactobacillus spp. with OB foals having more lactobacilli than CWB foals at 1 and 4 d (P < 0.05); however, there were no differences observed at 14 d (P > 0.05). Maternal diet did not influence amylolytic or cellulolytic bacteria (P > 0.05). These results indicate that colonization of the hindgut is a sequential process beginning early in the foal's life and that maternal diet may influence some bacteria in the gastrointestinal tract of foals.

Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo.

Cereal grains are often included in equine diets. When starch intake exceeds foregut digestion starch will reach the hindgut, impacting microbial ecology. Probiotics (e.g., lactobacilli) are reported to mitigate GI dysbioses in other species. This study was conducted to determine the effect of exogenous lactobacilli on pH and the growth of amylolytic and lactate-utilizing bacteria. Feces were collected from 3 mature geldings fed grass hay with access to pasture. Fecal microbes were harvested by differential centrifugation, washed, and re-suspended in anaerobic media containing ground corn, wheat, or oats at 1.6% (w/v) starch and one of five treatments: Control (substrate only), L. acidophilus, L. buchneri, L. reuteri, or an equal mixture of all three (107 cells/mL, final concentration). After 24 h of incubation (37°C, 160 rpm), samples were collected for pH and enumerations of total amylolytics, Group D Gram-positive cocci (GPC; Enterococci, Streptococci), lactobacilli, and lactate-utilizing bacteria. Enumeration data were log transformed prior to ANOVA (SAS, v. 9.3). Lactobacilli inhibited pH decline in corn and wheat fermentations (P < 0.0001). Specifically, addition of either L. reuteri or L. acidophilus was most effective at mitigating pH decline with both corn and wheat fermentation, in which the greatest acidification occurred (P < 0.05). Exogenous lactobacilli decreased amylolytics, while increasing lactate-utilizers in corn and wheat fermentations (P < 0.0001). In oat fermentations, L. acidophilus and L. reuteri inhibited pH decline and increased lactate-utilizers while decreasing amylolytics (P < 0.0001). For all substrates, L. reuteri additions (regardless of viability) had the lowest number of GPC and the highest number of lactobacilli and lactate-utilizers (P < 0.05). There were no additive effects when lactobacilli were mixed. Exogenous lactobacilli decreased the initial (first 8 h) rate of starch catalysis when wheat was the substrate, but did not decrease total (24 h) starch utilization in any case. These results indicate that exogenous lactobacilli can impact the microbial community and pH of cereal grain fermentations by equine fecal microflora ex vivo. Additionally, dead (autoclaved) exogenous lactobacilli had similar effects as live lactobacilli on fermentation. This latter result indicates that the mechanism by which lactobacilli impact other amylolytic bacteria is not simple resource competition.

Effect of Dietary Starch Source and Concentration on Equine Fecal Microbiota.

Starch from corn is less susceptible to equine small intestinal digestion than starch from oats, and starch that reaches the hindgut can be utilized by the microbiota. The objective of the current study was to examine the effects of starch source on equine fecal microbiota. Thirty horses were assigned to treatments: control (hay only), HC (high corn), HO (high oats), LC (low corn), LO (low oats), and LW (low pelleted wheat middlings). Horses received an all-forage diet (2 wk; d -14 to d -1) before the treatment diets (2 wk; d 1 to 14). Starch was introduced gradually so that horses received 50% of the assigned starch amount (high = 2 g starch/kg BW; low = 1 g starch/kg BW) by d 4 and 100% by d 11. Fecal samples were obtained at the end of the forage-only period (S0; d -2), and on d 6 (S1) and d 13 (S2) of the treatment period. Cellulolytics, lactobacilli, Group D Gram-positive cocci (GPC), lactate-utilizers and amylolytics were enumerated. Enumeration data were log transformed and analyzed by repeated measures ANOVA. There were sample day × treatment interactions (P < 0.0001) for all bacteria enumerated. Enumerations from control horses did not change during the sampling period (P > 0.05). All treatments except LO resulted in increased amylolytics and decreased cellulolytics, but the changes were larger in horses fed corn and wheat middlings (P < 0.05). Feeding oats resulted in increased lactobacilli and decreased GPC (P < 0.05), while corn had the opposite effects. LW had increased lactobacilli and GPC (P < 0.05). The predominant amylolytic isolates from HC, LC and LW on S2 were identified by 16S RNA gene sequencing as Enterococcus faecalis, but other species were found in oat fed horses. These results demonstrate that starch source can have a differential effect on the equine fecal microbiota.

Diarrhea-associated pathogens, lactobacilli and cellulolytic bacteria in equine feces: responses to antibiotic challenge.

Antibiotics are important to equine medicine, but antibiotic-associated diarrhea (AAD) can lead to poor performance and even mortality. AAD is attributed to disruption of the hindgut microbiota, which permits proliferation of pathogenic microbes. The goal of this study was to evaluate the effects of common antibiotics on cellulolytic bacteria, lactobacilli, and AAD-associated pathogens in the feces of healthy horses. Fifteen horses were assigned to three treatment groups (blocked by age and sex): control (no antibiotics), trimethoprim-sulfadiazine (PO), or ceftiofur (IM). Fecal samples (n=8 per horse) were taken during dietary adaptation (3 weeks), antibiotic challenge (1 week), and withdrawal (1 week). Bacteria were enumerated by serial dilution and viable count. Cellulolytic bacteria decreased by >99% during administration of either antibiotic (P<0.0001) and were still less than controls at the end of the withdrawal period (P<0.0001). Fecal samples from horses challenged with ceftiofur had 75% fewer lactobacilli than those from control horses at the end of the antibiotic challenge period (P<0.05). Antibiotic challenged horses also shed more salmonella than control horses (P<0.05). Antibiotics had no effect on the number of Clostridium perfringens isolates. There was no detectable Clostridium difficile during adaptation or in any control horse. C. difficile increased (P<0.0001) to approximately 10(4)cfu/g when horses were challenged with antibiotics, and were still detectable 1 week after withdrawal. These results indicate that antibiotics can disrupt the normal gastrointestinal microbiota and allow proliferation of Salmonella spp. and C. difficile.