The influence of hay steaming on clinical signs and airway immune response in severe asthmatic horses.

BACKGROUND: Avoidance of antigenic stimuli was found to significantly reverse airway obstruction of horses with severe equine asthma (sEA). To date, no published study investigated the influence of steaming hay on lower airway condition of sEA-affected horses. The objectives were to determine the clinical, cytological and cytokine respiratory responses of both sEA and control (CTL) horses experimentally exposed to steamed or dry hay. RESULTS: A cohort of 6 sEA horses and 6 CTL horses was involved in this field study. On day 0, both groups were fed with steamed hay for 5 consecutive days, followed by a wash-out period of 26 days prior to be fed with dry hay for 5 consecutive days. Investigations performed 2 days prior to and 5 days after each challenge included clinical score, tracheal mucus accumulation, and bronchoalveolar lavage fluid (BALF) cytology and cytokine mRNA expression. Feeding steamed hay significantly decreased its mould content (P < 0.001). Mucus score significantly increased when feeding dry hay (P = 0.01). No significant influence of challenge type was found on clinical score. Percentages of neutrophils (P < 0.001) as well as mRNA expression of IL-1ß (P = 0.024), IL-6R (P = 0.021), IL-18 (P = 0.009) and IL-23 (P = 0.036) in BALF of sEA affected horses were significantly increased after both (steamed and dry hay) challenges. Relative mRNA expression of IL-1ß, IL-6R and IL-23 in BALF were also significantly correlated to neutrophil percentages and both clinical and tracheal mucus score. CONCLUSIONS: Steaming significantly decreased mould content but inconsistently influenced the respiratory response of sEA affected horses when fed hay. Based on BALF cytology and cytokine profiles, its relevance might be controversial as a non-medicinal therapy for sEA-affected horses.

Where Are We Now? Feeds, Feeding Systems and Current Knowledge of UK Horse Owners When Feeding Haylage to Their Horses.

Despite our improved understanding of equid digestive health and accurate rations formulations, obesity in the UK horse population remains high. Study aims: (1) to determine how owners are feeding their horses and what influences their choices, (2) to understand owners' knowledge of haylage and (3) to identify key areas that require additional education. Data were collected in 2020 from 1338 UK horse owners via two online surveys. Survey 1 was on general feeding practices, and Survey 2 was specifically on the feeding of haylage. Data were processed using chi square analyses + Bonferroni tests, with a significance p < 0.05. Equal numbers of leisure and performance horse owners completed both surveys. For Survey 1, 67% fed hay as the only forage, 30% fed forage (hay/haylage) + balancer, 36% fed haylage and hay to manage energy intake, 84% added a cube or coarse mix, 88% did not do forage analyses, 74% did not see the need for it and 16% did not know analyses could be done. In Survey 2, those who were not feeding haylage, 66% were not sure how to feed it, 68% worried about aerobic spoilage and 79% said the bale size was unsuitable. Body weight measurements (Survey 1 and Survey 2) were rarely performed (11%). Aspects of ration formulations, the value of feed analyses and how to interchange hay and haylage require additional education to owners for improved ration compilation.

The haybiome: Characterising the viable bacterial community profile of four different hays for horses following different pre-feeding regimens.

Respirable dust in conserved forages can pose problems with equid respiratory health, thus soaking (W) and high temperature steaming (HTS) are employed to reduce the levels in hay. The aim of this study was to characterize the viable bacterial community profile of four hays from two different locations in UK following pre-feeding wetting regimens. Hypothesis: (1) Viable microbial community profile of hays will not differ between different pre-feeding regimens. (2) Hay type and location will not influence microbial community profile. Replicates of each of the four hays were subjected to dry (D), HTS conducted in a HG600, W by submergence in 45 L tap water, 16°C for 12 hours. From each post-treated hay, 100 g samples were chopped and half (n = 36) treated with Propidium monoazide dye, the remaining half untreated. Bacterial DNA were extracted for profiling the V4-V5 region of 16S rRNA gene from all 72 samples, then sequenced on the Illumina MiSeq platform. Bioinformatics were conducted using QIIME pipeline (v1.9.1). Linear discriminate analysis effect size (LEfSe) was used to identify differences in operational taxonomic units and predicted metabolic pathways between hays and regimens. HTS reduced proportions of microbiota compared to W and D hay (P < 0.001, df 3, F 13.91), viability was reduced within regimens (P = 0.017, df 1, F 5.73). Soaking reduced diversity within and between regimens. Core bacterial communities differed between hays and regimens, however pre-feeding regimen had the greatest effect on the bacterial community profile. W and HTS reduced viable bacteria (P< 0.05) known to cause respiratory disease, for HTS both respiratory and dental disease, with the greatest reductions overall from HTS without reducing bacterial diversity. Soaking increased Gram-negative bacteria and reduced bacterial diversity. Collectively these findings add to a body of evidence that suggest HTS is the most suitable pre-feeding regimen of hay for equid health.

The energy requirements of racehorses in training.

The aim of this study was to estimate the energy requirements of Thoroughbred racehorses in active training for flat racing. Twenty-two Thoroughbred racehorses in England were measured over periods from 6 to 15 wk, which included periods of active race training and temporarily reduced training. Energy intake was determined by measuring daily feed consumption. Energy output was measured using heart rate monitors during 730 training sessions, relating heart rate (HR) to oxygen consumption (VO2) and converting VO2 to energy. Field maintenance requirements were calculated by deducting the marginal energy cost of training from energy input. The mean field maintenance expenditure during periods of active race training was 0.1731 megajoules (MJ) of metabolizable energy (ME)/kg of bodyweight (BW)/d (SD = 0.0174, CI = 0.0073, n = 22 horses, 193 wk). This result is 11% to 66% greater than the official guidance found in the United States, France, Germany, and the Netherlands. Heart rate monitoring revealed a mean energy expenditure for exercise of 0.0212 MJ ME/d (SD = 0.0049, CI = 0.0007, n = 22 horses) for racehorses in active race training, a result 70% to 82% below the official guidance. The total mean energy expenditure for racehorses in active race training was 0.1943 MJ ME/kg/d (SD = 0.0177, CI = 0.0078, n = 20 horses 193 wk), 4% to 22% less than the official guidance. Horses actively racing had a 12% higher maintenance requirement than those in training but not yet racing (P = 0.01). The 2- and 3-yr-old horses did not gain weight during active race training, but grew slowly during breaks in training. This study explores the factors affecting energy balance in racehorses, and provides updated findings for their maintenance and training requirements.

The energy requirements of performance horses in training.

The aim of this study was to estimate the energy requirements of performance horses in active, variable training in the field. Sixty horses in England and Switzerland were measured over 2-wk periods and, for 15 of these, the measurement period was extended, ranging from 21 to 42 wk. Energy intake was estimated by measuring daily feed consumption. Energy output was measured using heart rate (HR) monitors during 608 training sessions, relating HR to volume of oxygen (VO2) and converting VO2 to energy. Field maintenance requirements were calculated by deducting the marginal energy cost of training from energy input. The mean field maintenance expenditure for performance horses with a normal temperament was found to be 0.118 MJ of metabolizable energy (ME) per kilogram of body weight (BW) per day (SD = 0.008, CI = 0.005, n = 60 horses). This result is between 1.9% (P = 0.086) and 20.9% (P < 0.001) greater than the official guidance found in the United States, France, Germany, and Holland. Heart rate monitoring of training revealed a mean energy expenditure (EE) per ridden session of 0.023 MJ ME (SD = 0.001, CI = 0.001, n = 175 training sessions). The mean daily EE for exercise based on a full week's training was 0.018 MJ ME/kg BW/d (SD = 0.005, CI = 0.001, n = 60 horses), representing a multiple of maintenance of 15.3%. This implies that the official guidance in the United States and France may overstate expenditure for exercise by 111% and 15%, respectively (P < 0.01). Daily EE between countries and within disciplines was consistent, allowing for the creation of user-friendly tables that can be used in budgeting the energy component of diets.

Growth Rates of Thoroughbred Foals and In Vitro Gut Health Parameters When Fed a Cereal or an All-Fiber Creep Feed.

Traditions and the economic advantage when producing big athletic Thoroughbred (TB) yearlings for sale encourage owners to feed high levels of cereals. Such diets can predispose gastric ulceration and developmental orthopedic diseases, impacting negatively on future careers. This study aimed to (1) determine if an all-fiber creep feed (total mixed fiber ration [TMFR] would sustain equal growth rates in TB foals versus a cereal-based stud cube (SC) and (2) compare pH and lactate produced by SC and TMFR in in vitro foregut and hindgut conditions. Eight TB foals in matched-pairs were fed SC or TMFR for 18 weeks. Weight gain (average daily gain [ADG]), height at wither and hip, heart girth, and body length were recorded weekly. Similar growth rates were recorded for all measurements. The ADG of TMFR and SC were 1 and 1.1 kg per head per day, respectively. In vitro foregut incubation of SC and TMFR at 37°C in pepsin HCl solution for 6 hours produced higher pH 5.84 (TMFR) compared with 5.33 (SC) (P < .05). Gas production measured fermentation rate, acidity, and lactate from TMFR and SC, incubated with fecal inoculum from the foals on the same diet. Fermentation rates and lag times were equal for both feeds; total gas produced at t50 and y50 were greater (P < .05) for SC. Lactate and pH levels were lower and higher, respectively, for the TMFR. This study showed that similar growth in TB foals was achieved on the TMFR feed and that potentially better gut health, denoted by higher pH and lower lactate levels, could be maintained by fiber compared with cereal feed.

Mapping the bacterial ecology on the phyllosphere of dry and post soaked grass hay for horses.

Soaking hay fodder to reduce dust and soluble carbohydrate (WSC) contents prior to feeding is common practice among horse owners. Soaking can increase bacteria load in hay but no information exists on how this process alters the bacteria profile, which could pose a health risk or digestive challenge, to horses by introducing foreign bacteria into the gastrointestinal tract and so altering the normal profile. The current objectives were to map the bacterial profile of 3 different hays and determine how soaking alters this with the aim of improving best practice when feeding stabled horses. A Perennial Rye grass hay and two meadow s hays were soaked for 0, 1.5, 9 or 16 hours. Pre and post treatment, hays were analysed for WSC and total aerobic bacteria (CFU/g), and differences in bacteria family profiles were determined using ANOVA with significance set at P<0.05. Bacteria were identified via genomic DNA extraction and 16S library preparation (V3 and V4 variable region of 16S rRNA) according to the Illumina protocol. Differences in family operational taxonomic units (OTUs) between individual dry hays and different soaking times were identified via paired t-tests on the DESeq2 normalised data and false discovery rates accounted for using Padj (P<0.05). Mean % WSC losses and actual g/kg lost on DM basis (+/- SE) increased with soaking time being 18% = 30 (10.7), 38% = 72 (43.7), and 42% = 80 (38.8) for 1.5, 9 and 16 hours soak respectively. No relationship existed between WSC leaching and bacteria growth or profile. Grass type influenced bacterial profiles and their responses to soaking, but no differences were seen in richness or Shannon diversity indices. PCA analyses showed clustering of bacteria between meadow hays which differed from the perennial rye grass hay and this difference increased post soaking. Soaking hay pre-feeding causes inconsistent WSC leaching, bacteria growth and alterations in bacterial profiles which are unpredictable but may decrease the hygienic quality of the fodder.

Development of a comprehensive protein microarray for immunoglobulin E profiling in horses with severe asthma.

BACKGROUND: Severe asthma in horses, known as severe equine asthma (SEA), is a prevalent, performance-limiting disease associated with increased allergen-specific immunoglobulin E (IgE) against a range of environmental aeroallergens. OBJECTIVE: To develop a protein microarray platform to profile IgE against a range of proven and novel environmental proteins in SEA-affected horses. ANIMALS: Six SEA-affected and 6 clinically healthy Warmblood performance horses. METHODS: Developed a protein microarray (n = 384) using protein extracts and purified proteins from a large number of families including pollen, bacteria, fungi, and arthropods associated with the horses, environment. Conditions were optimized and assessed for printing, incubation, immunolabeling, biological fluid source, concentration techniques, reproducibility, and specificity. RESULTS: This method identified a number of novel allergens, while also identifying an association between SEA and pollen sensitization. Immunolabeling methods confirmed the accuracy of a commercially available mouse anti-horse IgE 3H10 source (R2 = 0.91). Biological fluid source evaluation indicated that sera and bronchoalveolar lavage fluid (BALF) yielded the same specific IgE profile (average R2 = 0.75). Amicon centrifugal filters were found to be the most efficient technique for concentrating BALF for IgE analysis at 40-fold. Overnight incubation maintained the same sensitization profile while increasing sensitivity. Reproducibility was demonstrated (R2 = 0.97), as was specificity using protein inhibition assays. Arthropods, fungi, and pollens showed the greatest discrimination for SEA. CONCLUSIONS AND CLINICAL IMPORTANCE: We have established that protein microarrays can be used for large-scale IgE mapping of allergens associated with the environment of horses. This technology provides a sound platform for specific diagnosis, management, and treatment of SEA.

Neural modulators of temperament: A multivariate approach to personality trait identification in the horse.

A relationship between dopamine and temperament has previously been described in human cases of dopaminergic dysfunction. Adjustment in temperament prior to disease manifestation can enable the early identification of individuals at risk of such conditions, and scope exists to extend this application of temperament alterations to cases of dopaminergic dysfunction in horses. A multivariate and mixed-methods approach utilising a questionnaire along with two inferred measurements of dopamine activity (Spontaneous Blink Rate [SBR] and behavioral initiation rate [BIR]) were recorded from direct observation of animals (n=99) to identify the potential relationship between dopamine and temperament in horses. Principal components analysis (PCA) of 36 temperament variables revealed nine principal components, including 'Anxiety' and 'Docility', which accounted for 72.4% of the total variance. Component scores were calculated and correlated with SBR and BIR utilising Spearman rank correlation coefficient analysis. The component 'Anxiety' was found to have a significant positive relationship with SBR, whereas 'Docility' was observed to have a significant negative relationship with SBR. These results indicate a relationship between dopamine and temperament within the horse that is certainly worthy of further study. Potential mechanisms involving neural dopaminergic and GABAergic systems are presented, in addition to how such alterations could be utilised to probe for equine dopamine dysfunction pending future research.

The effect of five different wetting treatments on the nutrient content and microbial concentration in hay for horses.

Five different hays were used to determine the effect of 5 different soaking and steaming treatments on the water soluble carbohydrate and microbial (bacteria and mould) contents of UK hay. Hays were subjected to the following 5 treatments: 1. Dry; 2. Steamed for 50 minutes in the Haygain- 600 steamer; 3. Soaked in water at 16°C for 9 hours; 4. Steamed then soaked and 5. Soaked then steamed. Post treatment hays were tested for water soluble carbohydrates, bacteria and mould contents. Differences between means were determined using ANOVA and least significant difference with hay (5), bale (3) and treatment (5) as fixed factors, thus n = 75. Protein and ash proportions were unaltered in any of the treatments. Soaked, steamed then soaked and soaked then steamed treatments were all equally effective at reducing water soluble carbohydrates, with significantly (P<0.05) lower mean contents (79-83 g/kg DM) compared with 126 and 122 g/kg dry matter (DM) for dry and steamed respectively. Steamed and soaked then steamed had significantly (P<0.05) less bacteria (1.04×103 and 4.9×102 CFU/g DM) compared with soaked which increased CFU/g DM from 6.0×104 in dry hay up to 3.5×105. Mould contents CFU/g DM were significantly (P<0.05) reduced by steaming (2) and soaking then steaming (1.9) but no difference was seen between dry (1148), soaked (692) or steamed then soaked (501). Soaking for 9 hours followed by steaming for 50 minutes in the Haygain steamer was the most effective method for reducing water soluble carbohydrates and microbial contamination in hay. Soaking or steaming+soaking lowered water soluble carbohydrates but significantly reduced the hygienic quality of the hay which could potentially compromise the health of the horse.

The effect of enzyme treatment on the in vitro fermentation of lucerne incubated with equine faecal inocula.

A series of experiments was conducted to determine the effects of a fibrolytic enzyme preparation (enzyme 1; E1) on the in vitro fermentation of lucerne incubated with equine faecal inocula. In experiment 1, high-temperature-dried (HT) lucerne was treated with five levels of E1 (0 to 2.4 ml/g DM) and incubated at 50 degrees C for 20 h. Samples then received a simulated foregut digestion (SFD) treatment before DM and NSP analysis. In experiment 2, HT lucerne was treated with the same enzyme levels used in experiment 1. Samples were then split into two groups; plus or minus an SFD treatment before in vitro fermentation using an equine faecal inoculum. In experiment 3, fresh and wilted lucerne were treated with the same levels of E1 as experiments 1 and 2, incubated at 50 degrees C for 20 h, then fermented in vitro. For experiment 4, fresh and wilted lucerne were treated with low levels (0 to 0.008 ml/g DM) of E1 before fermentation. E1 significantly (P<0.05) enhanced DM and NSP losses from HT lucerne following SFD treatment compared with the control. High levels of E1 significantly (P<0.05) enhanced the rate, but not extent, of fermentation of HT, wilted and fresh lucerne; however, low levels of E1 were ineffective. At higher application levels, E1 appears to have considerable potential to enhance the nutritive value of lucerne for horses. Information on the fermentation kinetics of the substrates was valuable; all end-point measurements showed no effect of enzyme treatment.