Long-term effects of endurance training on total tract apparent digestibility, total mean retention time and faecal microbial ecosystem in competing Arabian horses
Reasons for performing study: In endurance horses, commonly fed 80% forage, energy is provided mainly by VFA produced in the hindgut during cell wall degradation, but cell wall digestibility has been reported to be impaired by exercise.
Objectives: To assess the influence of a long-term endurance conditioning on cell wall digestion in horses.
Methods: The total tract apparent digestibility of dry matter, organic matter and fibrous constituents, solid and liquid total mean retention time and the faecal microbial ecosystem of purebred Arabian horses were measured for 2 years in 2 longitudinal experiments.
Measurements: Performed at the beginning of each year for control level and after conditioning periods corresponding to incremental endurance racing levels: 60, 90 (year 1) and 120 (year 2) km. During the 5 measurement periods, feed intake and diet composition were similar.
Results: In year 1, digestibility of DM, OM and NDF was higher after 10 weeks of training (P = 0.008, P = 0.010 and P = 0.031, respectively), corresponding to the 90 km level, compared to the pretraining level. In year 2, NDFd and (NDF–ADF)d tended to be higher (P = 0.06 and P = 0.07, respectively) after the 17 weeks conditioning necessary to reach 120 km level, than before training. These variations were not systematically associated to a longer total MRT, neither to an increase in the microbial fibrolytic activity.
Conclusions: Digestibility of DM, OM and NDF appeared to be higher after endurance conditioning. Such an increase could be beneficial for endurance horses as it would provide more energy from forage degradation. Additional experiments are needed to elucidate the mechanisms, understand some contradictory results and investigate methodological aspects.
In man, ‘exercise’ can be defined as voluntary activation of skeletal muscle leading to short-term effects (for minutes or hours) while ‘physical activity’ is considered as repetitive exercise periods leading to long-term effects (for days, weeks, months or years) (Peters et al. 2001). Both exercise and physical activity have been reported to impact digestion: exercise can lead to gastrointestinal disorders (Gil et al. 1998; Gisolfi 2000), whereas physical activity has been reported to improve digestive physiology and health (Peters et al. 2001; Simren 2002).
Digestion has also been shown to be modified in working horses, but data are scarse and results controversial. In some cases, dry matter (DM), organic matter (OM) and crude fibre total tract apparent digestibility were reported to decrease in exercising horses (Pagan et al. 1998; Bergero et al. 2002). These changes may be due to shorter mean retention times (MRT) of solid particles (Pagan et al. 1998) and/or reduction of microbial activity in the hindgut (Dougal et al. 2005). Conversely, DM total tract apparent digestibility was shown to increase in exercising horses and this was partly explained by a longer MRT of liquid (Orton et al. 1985). Experimental conditions, particularly exercise regimen during measurements, may explain these contradictions as they can cause misconception between short-term effects of exercise and long-term adaptations through physical activity. In order to assess the short-term effects of exercise, horses must work during measurement periods, whereas to study the long-term effects of physical activity and training, horses need to stay at rest during measurements. Therefore, it is essential to separately explore the impact of exercise and physical activity. This will help clarify if athletic horses digest as sedentary horses and if data obtained from nutritional studies conducted in horses at maintenance are appropriate. Also, if digestion differs in athletic horses, it could impact energetic supply from feedstuffs. In endurance horses, the investigation of the impact of physical activity on digestion is crucial, particularly in terms of energy, a key point in their performance. As the ration of endurance horses is commonly based on 80% forages (Ralston 1988; Crandell 2005; Goachet 2006), the primary energy source is supplied by volatile fatty acids (VFA) provided through microbial fermentation in the hindgut.
The aim of the present study was to investigate the impact of a long-term endurance training on cell wall total tract apparent digestibility and its explanatory factors: total tract MRT, faecal microorganisms and their fibrolytic activity.
Materials and methods
Two longitudinal studies were conducted for 2 consecutive years. Measurements were done at the beginning of each year for control level (P01 and P02) and after conditioning periods corresponding to incremental endurance racing levels: 60, 90 (year 1) and 120 (year 2) km. P01 and P02 were set after 3 months of rest and P60, P90 and P120 were set during the second week following an endurance competition (Goachet et al. 2009a). Each measurement period included 9 days of dietary adjustment and 4 days of sampling (Goachet et al. 2009b).
In year 1, 8 purebred Arabian horses (8 ± 2-years-old) with a bodyweight (bwt) of 445 ± 13 kg and a body condition score (BCS) of 3.5 ± 0.4 (on a scale of 0–5 according to the method of INRA-HN-IE 1997) at the beginning of the experiment were used. In year 2, 7 Arabian horses (9 ± 2 years old) with a bwt of 448 ± 13 kg and a BCS of 3.5 ± 0.3 were used. Six of the 8 horses used in year 1 were also used in year 2.
During the 2 studies, horses were maintained in individual 4 ± 3.5 m free stalls, on artificial bedding (TIERWOHL1) and had access to sandy paddocks 7 h per day, except during the sampling periods, when they were maintained in their stall to facilitate the total MRT samplings. Horses were weighed and their BCS recorded weekly throughout the experiment. They were dewormed 3 times per year (ERAQUELL1; EQUIMAX2; PANACUR3) and vaccinated against flu and tetanus (GRIPIFFA4).
During the 2 years of study, horses were fed meadow hay which was from the same area and forage producer (average biochemical composition: 87.9% DM, 94.8% OM, 66.9% Neutral Detergent Fibre [NDF], 35.8% Acid Detergent Fibre [ADF] in year 1 and 86.8% DM, 92.8% OM, 65.2% NDF, 36.4% ADF in year 2) and commercial pelleted concentrate (Horse Prim ROYAL HORSE™ providing 89% DM, 91% OM, 11% CP, 2.2% fat, 23% starch, 38% NDF and 21% ADF). Hay and pellets were offered in 2 equal meals, at 8.00 and 17.00 and 10.00 and 16.00 h, respectively.
Apart from the measurement periods, horses received a daily allowance of approximately 10 kg DM of hay. The daily allowance of concentrate increased progressively from 1–2.4 kg DM with training in order to maintain optimal bwt and BCS. On average, the level of feed intake varied from 2.5–2.8% bwt. During the measurement periods, the total dry matter intake (DMI) was fixed at 1.7% bwt with a forage : concentrate ratio of 70:30 for all horses.
Horses underwent a regular endurance training programme. Exercises of 15–30 km outdoor rides, on varied grounds, were performed every second day and comprised 20% walk at 6 km/h, 70% trot at 13 km/h and 10% gallop at 20 km/h. Each ride began with a 20 min warm-up phase and ended with a 10 min cool down phase at walk. To prepare horses for 120 km races, this basal training was completed with 2 specific training sessions: one 90 km exercise at 10 km/h one month preceding the race and one 2 h gallop at 18 km/h on a racetrack performed 2 weeks before each race. To validate the training levels, every horse competed in official endurance events following the FFE (French Equestrian Federation) and FEI (International Equestrian Federation) rules. Every training session was registered for each horse. The length of the training period necessary to reach the different levels was determined individually and horses competed in endurance events when the trainer considered them to be physically ready.
Measurements and analytical methods
For total tract apparent digestibility determination, a partial collection of faeces was performed during the 4 consecutive days of each measurement period (Goachet et al. 2009b). Samples of 300 g of fresh faeces were taken from each horse by rectal sampling at 08.00 and 18.00 h every day. Faecal and feed samples, were dried in an air-forced oven at 75°C to constant weight for DM determination, and then ground to pass through a 1 mm screen. Faecal samples were then pooled for each horse and period. Feed and faecal samples were analysed for OM by incineration at 550°C for 5 h (71/250/CEE) and sequential analysis of Neutral Detergent Fibre (aNDFom), Acid Detergent Fibre (ADFom) and lignin(sa) (Van Soest et al. 1991).
For MRT measurements, a partial collection of faeces was performed during 3 consecutive days (Hyslop 2005; Goachet et al. 2009b). A single meal of Europium-labelled hay (60 g) and Ytterbium-labelled pellets (60 g) mixed with sugar (sucrose) to increase palatability was offered to each horse at 16.30 h on the first day of each measurement period. Labelling procedure of feeds was previously described by Drogoul et al. (2000). A Cr-EDTA solution (125 ml) was administered via a naso-oesophagal tube at 17.00 h. Faecal collection began at 20.00 h and a faecal sample of about 300 g was taken from the horse bedding every 2 h during the 3 following days. Samples were dried in an air-forced oven at 75°C to constant weight for DM determination and ground to pass through a 1 mm screen. Chromium (Cr), Ytterbium (Yb) and Europium (Eu) were solubilised for each sample in duplicate (Christian and Coup 1954 as modified by Siddons et al. 1985). The Cr, Yb and Eu concentrations were determined for each sample using an atomic absorption spectrometer (SpectrAA 300 Zeeman VARIAN) with a wavelength set at 357.87, 398.80 and 459.40 nm, respectively.
For microbial enumeration and activity measurements, a faecal sample was taken from the rectum at 13.00 h on Day 3 of each measurement period. A sub-sample for microbial counts was immediately placed in a sterile CO2-saturated flask, which was held at 38°C in a water bath between collection and inoculation. Total viable anaerobic bacteria and lactic acid-utilising bacteria counts were determined in anaerobic roll tubes with a modified nonselective medium (Leeddle and Hespell 1980; Julliand et al. 1999) and on a selective medium (Mackie and Heath 1979), respectively. Lactobacilli spp. and Streptococci spp. were cultured in Petri plates on Rogosa agar5 and on a bile-esculin-azide agar medium (BK158HA5), respectively. Cellulolytic bacteria were counted on a modified broth medium containing filter paper as the sole cellulolytic substrate (Halliwell and Bryant 1963; Baruc et al. 1983; Julliand et al. 1999). The concentration of cellulolytic bacteria was taken as the most probable number of Mac Grady (Hughes and Plantat 1983). A second sub-sample of faeces was filtered (Blutex 100 µm) and pH of the filtrate was measured with an electronic pH-meter (MP1207) immediately after collection. The filtered faeces were immediately frozen (-20°C) for determination of D- and L-lactate (1 ml) and VFA [1 ml together with a preservative (0.1 ml mixture of 5% H3PO4 + 1% HgCl2)]. L- and d-lactic acids were assayed with an enzymatic reaction procedure EnzyPlus8 and quantified by spectrophotometry at 340 nm (MRX revelation9. Total VFA, acetate (C2), propionate (C3) and butyrate (C4) concentrations were assayed by gas-liquid chromatography Gas chromatograph model 437A6 (Jouany 1982).
Total tract apparent digestibility coefficients of DM (DMd), OM (OMd), aNDFom (NDFd), ADFom (ADFd) (NDF–ADF) as considered as an estimation of hemicellulose (NDF–ADF)d were calculated from the concentration of lignin(sa) as a marker (Pys 2000; Santos et al. 2005; Goachet et al. 2009b) in feed and feces collected at 08.00 and 20.00 h: d(X)=[(Xd/Ld)−(Xf/Lf)]/(Xd/Ld), where Xd is the concentration of X in the diet, Ld is the concentration of lignin(sa) in the diet, Xf is the concentration of X in the faeces, Lf is the concentration of lignin(sa) in the faeces.
MRT of Yb-labelled pellets (pellets-MRT), Eu-labelled hay (hay-MRT) and Cr-EDTA (liquid-MRT) were calculated from the concentration of markers excreted as Thielemans et al. (1978):
where ti is the time (h) from the dosage of markers to the midpoint of the ith collection interval, ci is the concentration of the marker in the ith sample, (ti is the interval (h) between the 2 samplings and n is the number of samples.
Statistical analysis was performed with the GLM procedure of Statistical Analysis Systems Institute (Anon 2003). The effect of the conditioning was tested each year (P01 vs. P60 and P90 in year 1 and P02 vs. P120 in year 2) on the bwt, the BCS, the digestibility coefficients (DMd, OMd, NDFd, ADFd [NDF-ADF]d), the MRT (hay-MRT, pellets-MRT and liquid-MRT), the microbial counts and the biochemical parameters. The ‘horse’ effect was considered a random effect. The variable response (X) was analysed according to the following statistical model: Xij=µ+ ai+ bj+εij, where Xijk is the studied variable, µ the overall mean, ai the fixed effect of ith ‘conditioning’, bj the random effect of jth ‘horse’ and εij normally distributed error. Logarithmic transformations were performed on microbial counts before statistical analysis. Means were calculated for all variables and compared using linear contrast (pdiff option of SAS). Differences were considered statistically significant at P<0.05.
Horses, training and diet
In year 1, 7 horses were able to perform 60 and 90 km races. In year 2, 4 horses were able to perform a 120 km race. The training period necessary to reach the different levels was 4.6 ± 1.0, 10.7 ± 3.1 and 17.1 ± 8.8 weeks for 60, 90 and 120 km races respectively. It represented a total of 128 ± 37, 454 ± 37 and 783 ± 290 km to reach P60, P90 and P120, respectively. When horses were not able to reach the conditioning level, it was systematically due to punctual lame problems during training. In years 1 and 2, bwt and BCS were significantly higher at rest than after conditioning (Tables 1, 2), with the most important variations between P01 and P90 (P<0.0001) and P02 and P120 (P<0.0001). The chemical composition of the offered rations remained stable between the different measurement periods within each year and even between years 1 and 2 (Table 3).
Table 1. Bodyweight, Body Condition Score, total tract apparent digestibility, total MRT and faecal microbial ecosystem at Year 1
|Yb MRT (h)||–||25.8a||32.1b||0.8||0.003|
|Eu MRT (h)||–||25.8||30.4||1.4||0.079|
|Cr-EDTA MRT (h)||–||20.5a||25.3b||1.0||0.02|
|Total anaerobic bacteria (log10 cfu/g)||7.4||7.2||7.1||0.1||0.24|
|Lactate utilising bacteria (log10 cfu/g)||7.1||7.0||7.0||0.1||0.83|
|Lactobacilli (log10 cfu/g)||5.0a||6.1b||6.1b||0.2||0.0005|
|Streptococci (log10 cfu/g)||6.5||6.2||6.7||0.4||0.66|
|Cellulolytic bacteria (log10 cfu/g)||5.2||5.5||5.7||0.2||0.58|
|Total VFA (mmol/l)||30.7||37.5||51.3||6.6||0.10|
Table 2. Bodyweight, Body Condition Score, total tract apparent digestibility, total MRT and faecal microbial ecosystem at Year 2
|Yb MRT (h)||31.8||27.7||1.7||0.23|
|Eu MRT (h)||30.6||27.2||1.0||0.09|
|Cr-EDTA MRT (h)||26.5a||22.5b||0.8||0.04|
|Total anaerobic bacteria (log10 cfu/g)||7.7a||7.1b||0.1||0.01|
|Lactate utilising bacteria (log10 cfu/g)||7.6a||6.6b||0.1||0.01|
|Lactobacilli (log10 cfu/g)||5.9||5.8||0.1||0.51|
|Streptococci (log10 cfu/g)||6.4a||6.1b||0.4||0.04|
|Cellulolytic bacteria (log10 cfu/g)||5.9||6.1||0.3||0.71|
|Total VFA (mmol/l)||31.6||32.6||2.7||0.80|
Table 3. DM, OM, NDF, ADF, ADL and hemicelluloses concentrations of the whole diet (6.0 kg hay/2.4 kg concentrate) at each measurement period
Total apparent digestibility, total MRT and faecal ecosystem
In year 1, DMd, OMd and NDFd coefficients were higher at P90 than at P01 (P<0.05) (Table 1). Due to a technical problem at P01, we were not able to analyse the faeces for the total MRT determination. Pellets- and liquid-MRT were higher at P90 than at P60 (P<0.05). Among the variables characterising the faecal microbial ecosystem, only the pH and the lactobacilli and propionate concentrations varied between periods (P<0.01): pH was lower at P60 than at P01 and P90, lactobacilli were more numerous at P60 and P90 than at P01 and propionate concentration was higher at P90 than at P01 (Table 1).
In year 2, NDFd and (NDF–ADF)d tended to be higher at P120 than at P02 (P = 0.06 and P = 0.07) (Table 2). Liquid MRT, total anaerobic, lactate-utilising bacteria and Streptococci concentrations were significantly lower after a 120 km endurance conditioning than at rest (P<0.01) (Table 2). Other variables did not differ significantly between periods.
Our horses were easily able to perform 60 and 90 km races but only 4 had the capacity to participate in a 120 km race. The major limit for reaching these high levels of endurance racing was related to locomotion disorders, which corroborates the field observations in that discipline. It is considered that many horses are able to compete in 60 km endurance competitions, without a specific individual capacity, training or feeding programme. On the other hand, performing at 90 km level and above is more selective. Conditioning was linked to a decrease in bwt and BCS. After the 90 and 120 km training programme, the horses' BCS was 2.7, which is close to the recommendation of 2.5 (Geor 2005; Harris 2005) and which is considered by endurance horse trainers and riders to be the optimal BCS to perform in those competition levels in France (Goachet 2006). The results of our experimental horses in endurance racing as well as their fitness evolution illustrated that they received an appropriate training programme which enabled measurements to be made at incremental conditioning levels. This proved also that our research animals could be managed as elite endurance horses, in strictly controlled environmental conditions. The horses were maintained in similar housing and environment, received the same feeds at similar intake during the measurement periods, underwent the same training programme and also competed in official endurance events.
In year 1, digestibility was higher after a 90 km training programme, in particular DM (+6.7%-units), OM (+7.3%-units) and NDF (+7.6%-units) digestibility (Table 1). In year 2, digestibility coefficients increased numerically but not significantly at 120 km level; however, the statistical analysis was done with 4 horses and it could be assumed that with more individuals the differences for NDFd and (NDF–ADF)d would be greater. Indeed, this increase in digestibility after conditioning has been previously observed by Orton et al. (1985) and recently confirmed (Goachet et al. 2009c): in Arabian horses competing in 120 km endurance events, the digestibility of OM and cell-walls was significantly higher after 11 weeks of conditioning than before conditioning. In the present study, it should be noted, however, that digestibility of all nutrients was particularly high at P02 and surprisingly higher than at P01. For the 6 horses engaged in both experiments (years 1 and 2), the difference in digestibility ranged from 9.7–19.8% units between the 2 initial levels. Digestibility can be influenced by several factors, related to animals, diet and environment (Ragnarsson 2009). In our study, most of these factors were controlled. The analytical composition of the whole ration remained constant within each year. Regarding the length of our experiments, an hypothesis was that the increasing age of horses, as well as the season, could interfere with the effect of training. Age probably influences digestion either in very young (Cymbaluk et al. 1989; Julliand et al. 1996; Yuyama et al. 2004; Ringler et al. 2009) or mature horses, more than 20-years-old (Ralston et al. 1989). In mature horses, such an hypothesis has not been demonstrated and is questionable. Regarding season, an increase in DM digestibility has been associated with higher temperatures in ovine and bovine fed high fibre diet (Morand-Fehr and Doreau 2001). In our study, the control measurements (P01 and P02) were performed during winter whereas P60, P90 and P120 were performed in spring and summer. However, no potential explanation can be given to explain the differences between P01 and P02. Further investigations are planned in order to measure the reproductibility and repeatability of nutrient digestibility in horses.
An increase of cell wall digestibility could result from an increase of the fibrolytic activity of the microorganisms in the hindgut and/or an increase of the digesta retention time. A higher (C2+C4)/C3 ratio, reflecting a higher fibrolytic activity (Sauvant et al. 1994), could be expected but this did not occur. In year 1, the longer MRTs of both solid and liquid phases increased the duration of digesta exposure to enzymatic and microbial activity. This potentially explained the higher digestibility coefficients at P90. However, no conclusion can be drawn regarding the impact of conditioning on digesta passage rate as we were not able to compare with basal measurements at P01. Moreover, in year 2, MRT of liquid was shorter and MRT of labelled-hay tended to be shorter in trained horses. In man, even if the effects of physical activity are less known than those of exercise, some adaptations of the digestive system have been demonstrated (Gisolfi 2000). In particular, endurance training increase gastric emptying rate and increase gastrointestinal motility without any decrement in absorptive capacity. We can hypothesise that in horses as in human athletes, physical activity would influence gastric emptying and/or precaecal motility, more than the total MRT. This could partly explain the increase in amylolytic activity in the hindgut as shown by the higher faecal lactobacilli and propionate concentrations as well as the lower (C2+C4)/C3 ratio we observed in trained horses in year 1. This greater amylolytic activity could be related to a greater OM supply in large intestine. This may come from a more rapid precaecal passage rate.
In athletic horses, the only material that can be used to explore digesta passage rate and microbial activity is faeces, opposite to fistulated animals that give access to intestinal contents in different digestive segments. This limits the understanding of the mechanisms involved in digestion. Regarding passage rate, these constraints can be partially removed by using models to estimate digesta flow in different intestinal compartments (Moore-Colyer et al. 2003; Rosenfeld et al. 2006). Furthermore, the use of faeces to represent the microbial ecosystem of the hindgut is questionable. The faecal bacterial community seems to be different and more diverse than those present in the colon and caecum (Sadet-Bourgeteau et al. 2010). However, it has been suggested that the faecal ecosystem could be an appropriate marker of intestinal changes appearing in the colon (Julliand and Goachet 2005).
The present study showed that, in year 1, DM, OM and NDF digestibility was higher in at P90 than before training. This could be linked to the longer MRT observed at that training level, but not to the increase of the microbial fibrolytic activity in the hind-gut. However, these data were not confirmed in year 2 in horses trained to perform 120 km endurance events. Further experiments are needed to elucidate the interaction between training and digestion. If a positive long-term effect of endurance training is confirmed on digestibility, it could be beneficial for endurance horses as it would mean a higher energy yield from the digestion of feedstuffs and particularly from forage degradation. Moreover, it would raise the hypothesis that athletic horses digest differently than sedentary horses. This finding should then be taken into account when nutritional studies are conducted on horses at maintenance and results extrapolated to sport horses.
Authors acknowledge EVIALIS, Le Conseil Régional de Bourgogne et Le Fonds Social Européen for their financial support. They would like to thank T. Art and A. Breuvart for their scientific support, B. Chateau, P. Dorsemaine, E. Jacotot, A. Le Morvan, L. Martin, C. Sivry and M.C. de Vos Franzin and Dr J.M. Ricard, for their technical assistance. They are also very grateful to all the students who helped with training horses and to Marcel Mézy, the horses owner.
Conflicts of interest
1 Virbac, Switzerland.
2 Pfizer Animal Health, USA.
3 Internet Schering-Plough Animal Health, The Netherlands.
4 Merial, France.
5 Biokar Diagnostics, Beauvais, France.
6 United Technologies Packard, Zurich, Switzerland.
7 Mettler, Toledo, Spain.
8 D-L-Lactic Acid, Roma, Italy.
9 Dynatech Laboratories, Guyancourt, France.