Dietary supplementation of micro-encapsulated sodium butyrate in healthy horses: effect on gut histology and immunohistochemistry parameters.

BACKGROUND: As colic and intestinal disorders are a major concern in horses, the aim of the present study was to investigate the effect of dietary supplementation of butyrate, known to have a diverse array of beneficial effects on intestinal health. The effect of micro-encapsulated sodium butyrate supplementation on gut histology and immunohistochemistry parameters was studied in 14 healthy warmblood horses destined for slaughter in two separate periods. Horses were fed a low fiber - high starch diet, designed to induce subsequent starch overflow in the large intestine, aiming to create a mild challenge for large intestinal health. Treatment included supplementation with either micro-encapsulated sodium butyrate (Excential Butycoat®, Orffa, Werkendam, the Netherlands) or placebo (containing only coating material). The horses were fed for 20 consecutive days at a dosage of 0.4 g/kg BW (body weight). At day 21, the horses were slaughtered and intestinal samples were collected for determination of gut pH, villus length, crypt depth and area % of CD3+ and CD20+ cells. RESULTS: Horses on the butyrate supplemented diet had significantly reduced crypt depths in the right dorsal colon compared to placebo-fed horses (P < 0.001). However, a treatment x period interaction (P = 0.002) was discovered regarding this parameter, which could not be explained by the authors. Further investigation into the number of KI67+ cells in the RDC crypts did not reveal any significant differences between treatments (P = 0.650), indicating that the reduction in crypt depth in butyrate-fed horses could not be explained by a significant difference in cellular proliferation. Intestinal pH, villus length and expression of intestinal CD3+ and CD20+ cells were not significantly affected by treatment at any intestinal level. CONCLUSIONS: Our data indicate that supplementation of micro-encapsulated sodium butyrate to the equine diet did not influence gut histology (with the exception of a decrease found in the crypts of the RDC) or immunohistochemistry parameters in healthy horses. Further research is warranted to investigate the impact of butyrate supplementation in horses with intestinal disease.

Farming characteristics and self-reported health outcomes of Irish farmers.

Background: Irish farmers represent a 'high-risk' group for non-communicable diseases, which, arguably, pose a greater occupational health challenge for farmers. To date, there has been little exploration of the farming characteristics associated with farmers' poor health outcomes. Aims: To examine the relationship between farming and male farmers' self-reported health outcomes and to compare the study findings to national health studies to explore which factors specifically are associated with Irish farmers' poorer health outcomes relative to the general population. Methods: This cross-sectional survey research used self-reported quantitative data on the health outcomes and health behaviours of male farmers from the South-East of Ireland. Data were entered into SPSS and descriptive and binary regression techniques were used for data analysis. Results: There were 314 participants (99% response rate). Age, full-time farming and dairy farming significantly impacted self-reported health outcomes and health behaviours. There was a high prevalence of self-reported arthritis compared with the national average of Irish males. 'Younger' farmers (<45 years) were more likely to engage in harmful health behaviours such as smoking and 'binge-drinking' one or more times per week. Conclusions: This study identified self-reported patterns of risky lifestyle behaviours among particular subgroups of Irish farmers for whom targeted health interventions are warranted. Interventions are particularly important for younger farmers who may see themselves as invincible and impregnable to ill-health.

New insights into sequence variation in the IGS region of 21 cyathostomin species and the implication for molecular identification.

Cyathostomins comprise a group of 50 species of parasitic nematodes that infect equids. Ribosomal DNA sequences, in particular the intergenic spacer (IGS) region, have been utilized via several methodologies to identify pre-parasitic stages of the commonest species that affect horses. These methods rely on the availability of accurate sequence information for each species, as well as detailed knowledge of the levels of intra- and inter-specific variation. Here, the IGS DNA region was amplified and sequenced from 10 cyathostomin species for which sequence was not previously available. Also, additional IGS DNA sequences were generated from individual worms of 8 species already studied. Comparative analysis of these sequences revealed a greater range of intra-specific variation than previously reported (up to 23%); whilst the level of inter-specific variation (3-62%) was similar to that identified in earlier studies. The reverse line blot (RLB) method has been used to exploit the cyathostomin IGS DNA region for species identification. Here, we report validation of novel and existing DNA probes for identification of cyathostomins using this method and highlight their application in differentiating life-cycle stages such as third-stage larvae that cannot be identified to species by morphological means.

Influence of high phosphorus intake on salivary and plasma concentrations, and urinary phosphorus excretion in mature ponies.

This study addressed the question whether the concentration of phosphorus (P) in saliva of ponies is influenced by P intake. Six ponies were fed a diet high in P (HP treatment), providing 21 g P/day, and a diet low in P (LP treatment), supplying 7 g P/day. The two diets provided approximately 21 g calcium (Ca) and 6 g magnesium (Mg)/day. The experiment had an A-B-A design with treatment periods of 30 days. The ponies first received the HP diet (HP1), followed by the LP treatment and were then fed again the HP diet (HP2). Urinary P excretion was increased in both HP feeding periods and equalled approximately 7% of P intake vs. 0.5% on the LP diet. Plasma P concentration was higher for the HP treatment. The salivary P concentration ranged from 0 to 1.01 mmol P/l between ponies and there was no effect of P intake. It is suggested that saliva is not an important excretion route of P. The percentage of Ca and Mg in urine (% of intake) was higher for the LP treatment than for the HP treatments. The results of this study suggest that salivary Mg may contribute to Mg homeostasis.

Oocyst shedding patterns of Eimeria species and their association with management and performance at ten rose veal starter farms in the Netherlands.

Coccidiosis at rose veal starter farms is often diagnosed however, this was the first study performed considering this topic on this type of rearing unit. The objective of this study was to identify Eimeria species faecal shedding patterns at ten Dutch rose veal starter farms during rearing. Further objectives were to investigate associations with (gut) health, production and management decisions. Faecal samples from twelve randomly selected calves per farm were collected weekly during 9 consecutive weeks. Thereafter samples were pooled in a predetermined composition of six. These calves were clinically evaluated every sample visit and weighed at the first and last visit. Laboratory tests included a flotation test of the samples. If this yielded Eimeria oocysts, both oocysts per gram feces (OPG) and species differentiation were established using a modified McMaster method. Management parameters and technical herd results were identified after finalizing the study period using a questionnaire. Studied patterns in oocyst shedding included the pool's cumulated OPG, maximum OPG level and the number of sample days (SD) that OPG exceeded a confirmed level. Statistical analysis included univariate and multivariate analysis. Associated (p < 0.10) OPG patterns considering rumen fill, faecal consistency and average daily weight gain (ADG) were tested using a model with herd included as random effect. RESULTS: on all ten farms E. alabamensis, E.bovis and E. zuernii besides non-pathogenic species were identified, often as mixed infections. Peak OPG occurred predominately at SD 21 and 28 (E. alabamensis and E. bovis), and at SD 42 (E. zuernii). In 16 pools, OPG levels ≥500 for E. bovis or E. zuernii, were found. Significant correlation (p < 0.0001) showed between the log10 OPG of pathogenic Eimeria and of all Eimeria species. Multivariate analysis showed significant correlation between cumulative faecal consistency scores and cumulative log10 OPG of pathogenic Eimeria species (ß = 0.16; p = 0.008). Pools exceeding 750 OPG for E. bovis showed 93 g lower ADG and pools experiencing ≥2 SD with >1000 OPG for E. alabamensis 141 g lower ADG. From the questionnaire we identified lower cumulated OPG of all Eimeria species except for E. bovis at farms where the units were cleaned before arrival of the calves. CONCLUSION: As a rule, on Dutch rose veal starter farms, mixed Eimeria infections occur, but shedding patterns differ between farms. Clinical and growth performance is related to OPG patterns found. Cleaning units before arrival of calves lowers oocyst shedding during the rearing period.

Dynamic evaluation of toe-heel and medio-lateral load distribution and hoof landing patterns in sound, unshod Standardbred horses with toed-in, toed-out and normal hoof conformation.

Dynamic load distribution and landing patterns play an important role in equine orthopaedics. The aim of this study was to analyse dynamic load distribution and hoof landing patterns of sound Standardbreds. Twenty-four sound, unshod Standardbreds were walked and trotted over a pressure plate to obtain the peak vertical force (PVF), vertical impulse (VI) and stance time (ST) of both forelimbs. Asymmetry indices between both forelimbs were calculated and the dynamic medio-lateral and toe-heel load distribution of the forelimbs were compared between normal, toed-in and toed-out horses. The hoof landing patterns were determined on the first loaded zone. Linear mixed models and Chi-square analysis were used for statistical analysis (α = 0.05). At walk and trot, there were no significant differences in asymmetry of PVF, VI and ST between normal (n = 9/24 horses), toed-in (n = 6/24 horses), and toed-out horses (n = 9/24 horses). In both forelimbs, a flat landing (39.6% and 70.8% at walk and trot, respectively) predominated over all hoof conformations. No significant association between hoof conformation and landing pattern was observed (P > 0.05). Toed-out horses had significantly higher loading of the medial zone at the end of the stance phase at walk than normal horses (P = 0.002). No other statistically significant differences in load distribution were observed (P > 0.05). In conclusion, flat landing was more common in Standardbred horses than previously described in Warmbloods, emphasising the need for breed-specific clinical reference data and biomechanical studies. Moreover, mild deviations in hoof conformation did not necessarily affect load distribution.

The lipopolysaccharide model for the experimental induction of transient lameness and synovitis in Standardbred horses.

An established lipopolysaccharide (LPS) model previously described in Warmbloods, was inconsistent in Standardbred horses, where lameness was not detected despite the presence of synovitis. The present study aimed to determine the dose of LPS from E. coli O55:B5 required to induce mild to moderate lameness following middle carpal joint injection in Standardbred horses and to quantitate the induced lameness over time, with and without anti-inflammatory pre-treatment. In a baseline trial, eight healthy, clinically sound Standardbred horses were used in a rule-based dose-escalation design trial, starting at a dose of 10 endotoxin units (EU). Lameness at trot was evaluated visually and quantitatively (using an inertial-sensor system and pressure plate analysis). Synovial fluid aspirates were analysed for total nucleated cell counts, total protein and prostaglandin E2 (PGE2). Following 2 months wash-out, the effective LPS-dose determined in the baseline trial was used to evaluate the effect of anti-inflammatory treatment. A mixed model for repeated measures with horse as random effect was used for analysis. After injection of 10 EU LPS, the desired degree of lameness was observed in the baseline trial, with maximal lameness at post-injection hour (PIH) 4, followed by a rapid decline and return to baseline by PIH 48. No lameness was observed following pre-treatment with meloxicam. In synovial fluid, PGE2 was significantly higher at PIH 8 and PIH 24 in the baseline trial compared with following meloxicam pre-treatment. In conclusion, injection of the middle carpal joint with 10 EU LPS consistently induces a transient lameness and synovitis in Standardbred horses.

Domesticated equine species and their derived hybrids differ in their fecal microbiota.

BACKGROUND: Compared to horses and ponies, donkeys have increased degradation of dietary fiber. The longer total mean retention time of feed in the donkey gut has been proposed to be the basis of this, because of the increased time available for feed to be acted upon by enzymes and the gut microbiota. However, differences in terms of microbial concentrations and/or community composition in the hindgut may also underpin the increased degradation of fiber in donkeys. Therefore, a study was conducted to assess if differences existed between the fecal microbiota of pony, donkey and hybrids derived from them (i.e. pony × donkey) when fed the same forage diet. RESULTS: Fecal community composition of prokaryotes and anaerobic fungi significantly differed between equine types. The relative abundance of two bacterial genera was significantly higher in donkey compared to both pony and pony x donkey: Lachnoclostridium 10 and 'probable genus 10' from the Lachnospiraceae family. The relative abundance of Piromyces was significantly lower in donkey compared to pony × donkey, with pony not significantly differing from either of the other equine types. In contrast, the uncultivated genus SK3 was only found in donkey (4 of the 8 animals). The number of anaerobic fungal OTUs was also significantly higher in donkey than in the other two equine types, with no significant differences found between pony and pony × donkey. Equine types did not significantly differ with respect to prokaryotic alpha diversity, fecal dry matter content or fecal concentrations of bacteria, archaea and anaerobic fungi. CONCLUSIONS: Donkey fecal microbiota differed from that of both pony and pony × donkey. These differences related to a higher relative abundance and diversity of taxa with known, or speculated, roles in plant material degradation. These findings are consistent with the previously reported increased fiber degradation in donkeys compared to ponies, and suggest that the hindgut microbiota plays a role. This offers novel opportunities for pony and pony × donkey to extract more energy from dietary fiber via microbial mediated strategies. This could potentially decrease the need for energy dense feeds which are a risk factor for gut-mediated disease.

Multi-kingdom characterization of the core equine fecal microbiota based on multiple equine (sub)species.

BACKGROUND: Equine gut microbiology studies to date have primarily focused on horses and ponies, which represent only one of the eight extant equine species. This is despite asses and mules comprising almost half of the world's domesticated equines, and donkeys being superior to horses/ponies in their ability to degrade dietary fiber. Limited attention has also been given to commensal anaerobic fungi and archaea even though anaerobic fungi are potent fiber degrading organisms, the activity of which is enhanced by methanogenic archaea. Therefore, the objective of this study was to broaden the current knowledge of bacterial, anaerobic fungal and archaeal diversity of the equine fecal microbiota to multiple species of equines. Core taxa shared by all the equine fecal samples (n = 70) were determined and an overview given of the microbiota across different equine types (horse, donkey, horse × donkey and zebra). RESULTS: Equine type was associated with differences in both fecal microbial concentrations and community composition. Donkey was generally most distinct from the other equine types, with horse and zebra not differing. Despite this, a common bacterial core of eight OTUs (out of 2070) and 16 genus level groupings (out of 231) was found in all the fecal samples. This bacterial core represented a much larger proportion of the equine fecal microbiota than previously reported, primarily due to the detection of predominant core taxa belonging to the phyla Kiritimatiellaeota (formerly Verrucomicrobia subdivision 5) and Spirochaetes. The majority of the core bacterial taxa lack cultured representation. Archaea and anaerobic fungi were present in all animals, however, no core taxon was detected for either despite several taxa being prevalent and predominant. CONCLUSIONS: Whilst differences were observed between equine types, a core fecal microbiota existed across all the equines. This core was composed primarily of a few predominant bacterial taxa, the majority of which are novel and lack cultured representation. The lack of microbial cultures representing the predominant taxa needs to be addressed, as their availability is essential to gain fundamental knowledge of the microbial functions that underpin the equine hindgut ecosystem.

[Prevalence of Tritrichomonas foetus among Dutch cats]. / Prevalentie van Tritrichomonasfoetus onder Nederlandse katten.

Prevalence of Tritrichomonas foetus among Dutch cats The aim of this study was to determine the prevalence of and risk factors for, Tritrichomonas foetus among cats in the Netherlands. A total of 154 faecal samples were collected from three groups of cats: cats with diarrhoea (n=53), cattery cats (n=47), and healthy pet cats (n=54). Faecal samples were examined with a T. foetus specific real-time PCR. All PCR-positive samples were run on gel electrophoresis for definitive diagnosis. The prevalence of T. foetus was 2% among cats with diarrhoea and 4% among cattery cats; T. foetus was not prevalent among pet cats (none of the samples tested positive). Questionnaires had been distributed to cat and cattery owners to determine risk factors for T. foetus, but the low prevalence precluded statistical analysis of the questionnaire results.

Evaluation of titanium dioxide and chromic oxide as digestibility markers in ponies fed alfalfa hay in relation to marker dosing frequency.

In equines, Cr2O3 is widely accepted as an indigestible marker, but there are health concerns regarding the carcinogenic properties of Cr2O3. Recently, TiO2 has been suggested to be an alternative digestibility marker in equines. However, a comparison between Cr2O3 and TiO2 has not been made in equines. Six Welsh pony geldings (initial BW: 254±3 kg; 7 years of age) fed chopped alfalfa hay were used to evaluate the use of TiO2 (Ti) and Cr2O3 (Cr) as markers for calculating apparent digestibility and to investigate the effect of frequency of marker administration on the measurement of digestibility values. Diets contained 4.65 kg dry matter (DM) chopped alfalfa hay supplemented with minerals, vitamins, TiO2 (3.3 g Ti/day) and Cr2O3 (3.2 g Cr/day). Ponies were dosed with either 3.3 g Ti and 3.2 g Cr once daily (DF1) or with 1.65 g Ti and 1.60 g Cr twice daily (DF2). After adaptation to the diets and procedures for 14 days, voluntary voided faeces were collected quantitatively over 7 days and analysed for moisture, ash, Ti and Cr. Apparent total tract DM digestibility (DMD) and organic matter digestibility (OMD) were calculated using the total faecal collection (TFC) and marker method (Ti and Cr). The overall mean cumulative faecal recovery of Cr and Ti (as % of intake) were 102.0% and 96.6%, respectively. Mean daily faecal recoveries of Cr as well as of Ti were not different (P=0.323; P=0.808, respectively) between treatments. Overall daily faecal recovery of Cr differed (P=0.019) from 100% when the marker was dosed once daily, whereas overall daily faecal recovery was similar to 100% for both administration frequencies when Ti was used as a marker. For both markers, the coefficient of variation of the mean faecal marker recovery between horses was lower when the markers were administrated twice per day. Across treatments, cumulative DMD and OMD estimated with Ti were similar (P=0.345; P=0.418, respectively) compared with those values determined by TFC method. When Cr was used, the calculated cumulative DMD tended (P=0.097) to be greater compared with those estimated with TFC, and cumulative OMD values were overestimated (P=0.013). Orally supplemented Ti recovery in the faeces of ponies fed chopped alfalfa hay with Ti administered once or twice daily was close to 100%, making it the preferred marker for digestibility trials in equines.

The use of age-clustered pooled faecal samples for monitoring worm control in horses.

A study was performed on two horse farms to evaluate the use of age-clustered pooled faecal samples for monitoring worm control in horses. In total 109 horses, 57 on farm A and 52 on farm B, were monitored at weekly intervals between 6 and 14 weeks after ivermectin treatment. This was performed through pooled faecal samples of pools of up to 10 horses of the groups 'yearlings' (both farms), '2-year-old' (two pools in farm A), '3-year-old' (farm A) and adult horses (four pools on farm A and five pools on farm B), which were compared with the mean individual faecal egg counts of the same pools. A very high correlation between the faecal egg counts in pooled samples and the mean faecal egg counts was seen and also between the faecal egg counts in pooled samples and larval counts from pooled faecal larval cultures. Faecal egg counts increased more rapidly in yearlings and 2-year-old horses than in older horses. This implied that in these groups of young animals faecal egg counts of more than 200 EPG were reached at or just after the egg reappearance period (ERP) of 8 weeks that is usually indicated for ivermectin. This probably means that, certainly under intensive conditions, repeated treatment at this ERP is warranted in these young animals, with or without monitoring through faecal examination. A different situation is seen in adult animals. Based on the mean faecal egg counts on both farms and on the results of pooled samples in farm A, using 100 EPG as threshold, no justification for treatment was seen throughout the experimental period. However, on farm B values of 100 EPG were seen at 9 and 11, 13 and 14 and 14 weeks after ivermectin treatment in pools 10, 12 and 13, respectively. This coincided with the presence of one or two horses with egg counts above 200 EPG. The conclusion is that random pooled faecal samples of 10 adult horses from a larger herd, starting at the ERP and repeating it at, for instance, 4-week intervals, could be used for decisions on worm control. However, there would be a certain risk for underestimating pasture contamination through missing high-egg excreters. An alternative use of pooled samples would be as a cheap first screening to detect which adult horses really contribute to pasture contamination with worm eggs on a farm. All horses should be sampled and subsequently animals from 'positive' pools can be reexamined individually.

Pitfalls and opportunities of teaching veterinary parasitology within an integrated curriculum.

The Faculty of Veterinary Medicine at Utrecht University has seen three major curriculum changes, in 1995, 2001 and 2007. The last change was made because of the European change to a Bachelor-Master system. Almost each time teaching hours tagged for veterinary parasitology have been reduced to currently a minimum of between 46 and 51.5 h, which is much less than the WAAVP-recommended minimum of 70-90 h. This results in a challenge to maintain a qualitatively adequate veterinary parasitology program in a curriculum. Following a brief historic account of previous curricula and implemented curriculum changes, experiences, limitations and opportunities are discussed, including the potential of introducing new teaching materials based, for example, on digital technologies and gaming.

Equine parasite control and the role of national legislation - A multinational questionnaire survey.

Experts recommend a change in the control of equine parasites across the world in order to adopt a surveillance-based approach utilizing parasite faecal egg counts (FEC). Several European countries have implemented prescription-only restrictions of anthelmintic usage by law, which is in stark contrast to US, where all anthelmintic products continue to be available over the counter. This study aimed to describe and compare equine parasite control strategies employed in Germany, Austria, the Netherlands, US, and Denmark. An invitation to participate in an online questionnaire survey was published on a large equine news website in each of the participating countries. The main focus of the study was on usage of FEC and anthelmintic treatment intensity in three different equine age groups; foals, 1-3 year-olds and >3 years old. A total of 3092 respondents participated in the study. Danish respondents used significantly more faecal analyses in their parasite control strategies than participants from the other four countries (p < 0.0001). Similarly, Danish participants administered significantly fewer anthelmintic treatments per horse per year (p < 0.0001) independent of the age of the horse, and had been using a selective treatment strategy for a significantly longer time period than their counterparts in other countries (p < 0.0001). Only minor differences were found between respondents from the other four countries. This is remarkable as both Austria and Germany have had prescription-only restrictions for 3-4 decades. Yet, their parasite control strategies were more similar to those employed by American respondents. The Netherlands had only recently introduced prescription-only restrictions by the time this survey was conducted which can explain why Dutch respondents were also similar to their American counterparts. Taken together, this study illustrates substantially different worm control strategies practiced in Denmark, and this difference cannot be explained by legislation alone.

Ultrasonographic measurements of localized fat accumulation in Shetland pony mares fed a normal v. a high energy diet for 2 years.

Health risks associated with obesity are more likely a factor of the localization of fat excess, rather than of elevated BW per se. The aim of this randomized controlled clinical trial was to determine the effect of a long-term high energy diet on BW, fat accumulation and localization. Eight Shetland pony mares, 3 to 7 years old, were randomly divided into a control and a high energy (HE) diet group fed either maintenance or double maintenance energy requirements (200% net energy (NE)) for two consecutive summers, with a low energy diet in the winter in between. Body condition score (BCS) did not differ between the groups at the onset of the study (control 5.6±0.75 v. HE 6.3±0.5). From 12 weeks after starting the diet, ultrasonography of five different locations (retroperitoneal, axillary, withers, intercostal and rump) for adipose deposition, BCS and BW were measured monthly during the period that ponies received different diets. Statistical analysis was performed using a linear mixed-effects model with post hoc Bonferroni testing. P values <0.05 were considered significant. At week 12 after the onset of the diet, fat thickness in the HE group was significantly greater than in the control group. During the monitoring period, the HE group showed a significant increase in mean (±SE) BW (+52%, 265±13.94kg) and BCS (+70%; to 9.0±0.4), while the control group was unchanged (BW 160±13.98 kg; BCS 3.8±0.4). At all locations, the fat depth in the HE group increased significantly, with the highest increase noted for retroperitoneal deposits. The conclusions were that a 200% NE diet induced subcutaneous and retroperitoneal fat accumulation, with the greatest increase in intra-abdominal deposits. The moderate increase of the subcutaneous fat depth followed by a plateau phase suggests the existence of a limit of adipose tissue expandability, as in man.

Effect of exercise on apparent total tract digestibility of nutrients and faecal recovery of ADL and TiO2 in ponies.

Exercise and physical training are known to affect gastrointestinal function and digestibility in horses and can lead to inaccurate estimates of nutrient and energy digestibility when markers are used. The effect of exercise on apparent nutrient digestibility and faecal recoveries of ADL and TiO2 was studied in six Welsh pony geldings subjected to either a low- (LI) or high-intensity (HI) exercise regime according to a cross-over design. Ponies performing LI exercise were walked once per day for 45 min in a horse walker (5 km/h) for 47 consecutive days. Ponies submitted to HI exercise were gradually trained for the same 47 days according a standardized protocol. Throughout the experiment, the ponies received a fixed level of feed and the daily rations consisted of 4.7 kg DM of grass hay and 0.95 kg DM of concentrate. The diet was supplemented with minerals, vitamins and TiO2 (3.0 g Ti/day). Total tract digestibility of DM, organic matter (OM), CP, crude fat, NDF, ADF, starch, sugar and energy was determined with the total faeces collection (TFC) method. In addition, DM and OM digestibility was estimated using internal ADL and the externally supplemented Ti as markers. Urine was collected on the final 2 days of each experimental period. Exercise did not affect apparent digestibility of CP, crude fat, starch and sugar. Digestibility of DM (DMD), OM (OMD), ADF and NDF tended to be lower and DE was decreased when ponies received the HI exercise regime. For all treatments combined, mean faecal recoveries of ADL and Ti were 87.8±1.7% and 99.3±1.7%, respectively. Ti was not detected in the urine, indicating that intestinal integrity was maintained with exercise. Dry matter digestibility estimated with the TFC, ADL and Ti for ponies subjected to LI exercise were 66.3%, 60.3% and 64.8%, respectively, while DMD for HI ponies were 64.2%, 60.3% and 65.2%, respectively. In conclusion, physical exercise has an influence on the GE digestibility of the feed in ponies provided with equivalent levels of feed intake. In addition, the two markers used for estimating apparent DMD and OMD indicate that externally supplemented Ti is a suitable marker to determine digestibility of nutrients in horses performing exercise unlike dietary ADL.