Evaluation of urinary and faecal biological matrices highlighted significant metabolite profiles associated with an increase in exercise rigor within one yard. Horses from yard 3 experienced a gradual increment in exercise throughout the sampling period from the beginning of the training season to midway through. Therefore, these horses were unfit at the beginning of the sampling period, but had worked up to ¾ of their optimal speed within the last weeks of sampling (weeks 8-10). Unlike intensive race conditions, these animals were not pushed into long periods of anaerobic respiration. Anaerobic respiration occurs without sufficient oxygen to the muscles and therefore occurs when the energy demands of the horse are greater than can be supplied by the oxygen contained in blood.
Urinary p-cresol glucuronide (PCG) significantly reduced during the training period, while faecal sugars increased and to a lesser extent SCFAs. This may be a reflection of host and gut
PhD Thesis Ebony Ellen Escalona 166 microbiota energy requirements. PCG is produced by bacterial degradation of tyrosine, whereas SCFAs and sugars are produced by degradation of complex carbohydrates. This may reflect a switch or preference in carbohydrate metabolism over protein metabolism by the intestinal microbiome to keep up with energy demands of the host. Exercise may improve fibrolytic activity of the microbiota (Goachet et al., 2010) and a study by Dougal et al., 2005 showed a trend towards high counts of both total and cellulolytic bacteria (Dougal et al., 2005). A subsequent study by Goachet et al., described the effects of a five week exercise regime on previously inactive horses on faecal fermentative parameters. Volatile fatty acids were significantly higher after the 5 weeks exercise regime when compared to the dietary acclimatised starting period and suggests an increase in hindgut fermentation (Goachet et al., 2014).
Exercise is known to exert an effect on the metabolome of humans as acute physical workout will produce a plethora of metabolic changes in the body, altering lipid, bone and muscle metabolism (Suzuki et al., 1998; Hagg et al., 2005). However, many factors influence the metabolic signatures associated with exercise, such as gender, the fitness level of individuals, the intensity and duration of exercise undertaken and the time samples are taken with respect to completing exercise tests. The majority of metabonomic studies focus on blood samples, with very few if any assessing urinary or faecal biological matrices. A study by Enea et al., assessing urinary metabolites in trained and untrained female athletes revealed a number of energy metabolites associated with both intense short term exercise tests and prolonged exercise test after standardised urine collection (Enea et al., 2010). Other studies have also reiterated these findings (Pechlivanis et al., 2010). The metabolites identified by these studies include pyruvate, lactate and creatinine. In this equine study these energy metabolites were not identified as significantly associated with exercise in the cohort of young racehorses in training using supervised multivariate methods. This may be due to the lack of urine
PhD Thesis Ebony Ellen Escalona 167 collection standardisation and the relatively ‘easy’ exercise regime. However, univariate analysis of lactate levels in faeces did significantly increase with exercise and is shown in Figure 4.13B. Associated changes in tissue oxygenation can be seen in an increase in the lactate/pyruvate ratio which can be used as a crude marker for the degree of tissue oxygenation. An increase in this ratio reflects oxidative stress (Freikman et al., 2008) and in this study of Tb racehorses the mean lactate/pyruvate ratio of faecal samples more than doubled in the second half of the sample period compared to the first half (5.53 versus 16.98). As well as influencing intestinal physiology, exercise may also play a part in altering bacterial composition in the gut and therefore the potential to alter bacteria products. Studies have explored the effect of exercise on the gut microbial community in murine and human studies. A common finding among these studies is an increase in bacterial diversity in exercising subjects over sedentary controls. This has been shown in male rugby players
versus two control groups to match for body mass index (BMI) and age (Clarke et al., 2014)
and with male rats under differing dietary and physical exercise states (Isabel Queipo-Ortuno
et al., 2013). However, these studies only collected and analysed samples from one time point
and after minimal training times. There are very few studies addressing the effect of long- term exercise on gut microbiota or analysing biofluids from serial sampling. A study by Matsumoto et al., 2008 harvested murine caecal samples after a 5 week exercise period and highlighted changes in gut microbial metabolites and composition as well as caecal size and fill when compared to sedentary controls. These findings show the effects of ‘training’ on the gastrointestinal tract and unlike many other studies looking at exercise, dietary factors were standardised between case and control groups (Matsumoto et al., 2008). Goachet et al., 2010 also produced a study assessing the long term effects of exercise on faecal metabolites and microbiota in endurance horses over a 2 year sample period. Faecal propionate levels assessed via gas chromatography mass spectrometry (GC-MS) were found to be significantly
PhD Thesis Ebony Ellen Escalona 168 greater after training for 90km races than before conditioning. An increase in faecal propionate in response to training has also been demonstrated in this work. The linear increase in propionate is shown in Figure 4.13A.
Conclusions relating to exercise in this work should be interpreted with care, as this study was not specifically designed to explore exercise as a source of variation and specific details on the exercise regime employed in these horses is not known. However, these results do serve to act as pilot data to further investigate the effects of training on the Thoroughbred racehorse and in particular the gut microbiota. Gut microbial products not only tell us about gut microbial activity but these compounds may have important physiological functions far reaching from the intestinal site of origin. These results as well as others in the human literature show the utility of metabonomics within exercise performance. This novel form of information retrieval could soon supersede current physical activity monitoring tools such as blood pressure and heart rate measurements. Having a greater understanding on the performance capacity of a given Thoroughbred racehorse would be incredibly beneficial to trainers. Many trainers operate training regimes on cohorts of animals rather than on an individual basis and metabonomics offers the ability to obtain clinically valuable information on the response of exercise by an individual. Using this knowledge, refinement of training strategies could be made to ensure optimal health and athletic performance, having both beneficial outcomes for trainers and horses alike.
4.5.5 Urine metabolites correlate to a commonly used blood marker of over- training in Thoroughbred racehorses
GGT is a liver enzyme and is involved in glutathione metabolism and the transfer of glutamyl groups and although used as a liver function marker it also appears to be induced with exercise. The mechanism by which this occurs is unknown but levels of this enzyme have been shown to increase in response to training (Tyler-Mcgowan et al., 1999).
PhD Thesis Ebony Ellen Escalona 169 Clinical chemistry parameters were routinely taken from these horses as part of their health surveillance strategies to maintain performance levels. Two other blood markers, CK and AST were also measured but had no statistical correlation with 1H-NMR spectroscopic data from urine, plasma or faecal biofluids using multivariate data analysis. Perturbations in the level of this hepatic enzyme have also been associated with a number of intestinal pathologies, including colon displacements and proximal enteritis (Davis et al., 2003; Gardner et al., 2005) and the proposed mechanisms for this association include absorption of inflammatory mediators from the portal circulation and physical bile duct obstruction causing hepatic injury.
Intestinal links were also identified in this study by the positive correlation of urinary gut microbial co-metabolite dimethyl sulfone with blood GGT levels. Hydroxyphenyllactate was also significantly associated with GGT levels but was negatively correlated. Dimethyl sulfone is found in sulphur rich dietary plants and can be produced by endogenous and gut bacterial metabolism. It is an oxidation product of dimethyl sulfoxide and has been previously associated as a biomarker for onion intake (Winning et al., 2009). Hydroxyphenyllactate is formed from tyrosine and through gut bacterial action of dietary phenolic compounds. Low molecular weight phenolic acids such as hydroxyphenyllactate have been proposed to be involved in the regulation of the inflammatory response by reducing the production of reactive oxygen species in mitochondria and neutrophils from rat tissues. These findings suggest that hydroxyphenyllactate could have a protective effect on organs and tissues, acting as a natural antioxidant (Beloborodova et al., 2012). Bacteria associated with the production of hydroxyphenyllactate include Bifidobacteria and lactobacilli, which have been shown to produce considerable amounts of hydroxyphenyllactate via gas chromatography spectrometry. Despite GGT being used routinely as a marker of overtraining there was no association with GGT and exercise in this
PhD Thesis Ebony Ellen Escalona 170 population of Thoroughbred racehorses that had undergone incremental increase in exercise intensity. This is likely to be due to careful exercise management at the beginning of the training season that avoids overexertion. The mean GGT value in these horses was 18.5iu/l. Values commonly associated with overtraining are as high as 100iu/l (Snow and Harris, 1988).