**3. Discussion**

The results showed in the present paper highlight for the first time the changes occurring in the metabolomic profile of trained standardbred horses following exercise in both serum and saliva specimens.

Biochemical constituents of the tricarboxylic acid cycle (TCA or Krebs cycle) including succinate and fumarate were significantly increased in serum after exercise. This is in agreemen<sup>t</sup> with results obtained by other authors in human and equine athletes [25,29,30] and could be explained by the need to maintain and/or increase the Krebs cycle flow during exercise [8]. On the contrary, succinate and fumarate, together with malate, another constituent of the TCA cycle, have been found to decrease in saliva after exercise. Among salivary metabolites, malonate was found to have a very strong inhibitory effect on the TCA cycle in complex metabolic systems. Malonate is not normally present in cells [31] and the reason for its presence in saliva, its decrease after exercise and its inhibitory e ffect on the TCA cycle in horses during exercise need further investigations. Notwithstanding the paucity of data on salivary metabolomics during exercise, we can speculate that the decreased salivary level of succinate, fumarate and malate might reflect the imbalance of the aerobic pathways in favor of the anaerobic production of energy during exercise.

The increase in pyruvate and lactate serum concentrations was expected, since it reflects the anaerobic pathway activation during exercise [8,25]. The high energy demand required during exercise can overload the mitochondria's ability to oxidize pyruvate that is converted to lactate, supplying energy by the Cori cycle [8]. In our study, serum lactate increased up to 11.6-fold following exercise, and this result was beyond the physiological range usually reported for human athletes of a 9.3–9.6-fold increase [8,32]. Probably, the greater muscle mass of horses with respect to humans can account for this di fference. Although not significant, pyruvate and lactate slightly decreased in saliva after exercise (Supplemental Materials Table S1), demonstrating once again that salivary metabolic changes do not follow the same metabolic changes observed in serum, at least regarding the energy supply.

Among the metabolites detected in plasma, there are also two carbohydrates: mannose and arabinose.

The concentration of mannose resulted more concentrated in post-exercise equine plasma. This monosaccharide is found in small amounts in the diet, and after conversion to fructose-6-phosphate, it can be used in both glycolysis and gluconeogenesis [33]. Unlike mannose, the concentration of arabinose in serum appeared as decreased by exercise. Arabinose is known to have an inhibitory e ffect on intestinal sucrase [34], but its role in horse during exercise in not clear.

Galactose was the only carbohydrate with a changed concentration in saliva, as it decreased at T1. In a study on soccer players, Pitti et al. [21] found an increased level of galactose in blood after exercise. Galactose is an important source of glucose and its role in liver glycogen restoration after exercise has been well recognized in cyclists [33,35]. The reason for galactose reduction in the saliva of horses could reflect a di fferent energetic role of this metabolite compared with humans.

Among amino acids, alanine and histidine increased significantly after exercise. Serum alanine increased 1.6-fold soon after exercise, in agreemen<sup>t</sup> with the results obtained in humans by Berton et al. [8] but in contrast with Nieman et al. [29]. Alanine in muscle derives from pyruvate, in turn originated from glucose breakdown [8]. This metabolic mechanism is in line with the rise in serum pyruvate found in our study. Furthermore, alanine has a detoxing function by transporting to the liver the large amount of ammonia typically produced during short and high-intensity exercise derived from branched chain amino acids [8,36]. Similarly, the increase in gluconeogenic amino acid histidine could be linked to the higher energetic demand during acute exercise.

The decreased levels of glutamine, asparagine and proline observed in horses after training were similar to the findings in human athletes [29,30] thus supporting the hypothesis of an enhanced muscle amino acid oxidation during exercise to sustain the energetic demand.

In equine saliva, amino acids like tyrosine and phenylalanine rose almost 2-fold after exercise. Tyrosine results from hydroxylation of phenylalanine, an essential amino acid [37], and it is metabolized

to acetoacetate and fumarate or used as a precursor of catecholamines that play an important role in athletic performance during exercise [38]. We can speculate that the rise in salivary tyrosine after exercise could reflect an increased production to encounter the need for catecholamines during exercise, but the lack of similar changes in serum needs to be clarified.

Sarcosine (N-methylglycine) is a non-proteinogenic amino acid derivative and occurs in the body as a product of the metabolism of glycine and creatine [39]. In addition to its multiple functions in the body and its use as a potential marker in various diseases, it has been an ingredient in toothpaste for decades as it prevents tooth decay and causes foaming [39]. The reason for the slight rising of sarcosine and creatine in equine saliva after exercise is not clear, but its physiological function could be protective for teeth health.

Ornithine levels increased up to 2.1-fold in saliva but not in serum, di fferently from what has been observed in human athletes where it is down regulated 60–70 min after exercise as a consequence of the accelerated urea cycle due to the higher production of ammonia [8,30]. However, the di fferences in the sampling times of our study compared with others might explain this discrepancy. Betaine, a small molecule acting as a methyl donor in minor pathways [37], decreased in saliva after exercise, but this finding needs further investigations.

The marked elevation in serum glycerol in our horses following exercise resulted from an extensive lipolysis and is consistent with the study by Lewis et al. [30], who found higher serum glycerol up-regulation after exercise in fitter athletes. Considering that all the horses included in our study were well trained, we could consider glycerol a marker of adaptation to training also in equine species.

Salivary methanol was reduced 1.8 times by exercise. The same result was recently found in the saliva of soccer players, and the change was ascribed to the higher evaporation of volatile compounds in the mouth during exercise [21]. In a recent study, methanol was considered a marker of inflammation in horses a ffected by equine asthma when detected in exhaled breath condensate (EBC) [40].

Uracil nucleotide, together with cytosine, are the major pyrimidine components of RNA. Uracil can be utilized in glycogen synthesis [41] and its rise in saliva could be related to the higher energetic demand during exercise.

2-hydroxyisobutyrate was up-regulated in the serum of standardbred horses following exercise. This metabolite is a normal constituent of human serum and saliva [42] that has been proposed as a biomarker for glycogen storage disease type 1a in juveniles and acute coronary syndrome [43,44]. It was also found decreased in plasma of Alzheimer's disease patients vs. controls [45], however no information is available about its role during sport activity.

3-hydroxybutyrate decreased in serum after exercise. An elevation of this ketone was documented in marathon runners due to ketone production [30]. It was also found to be increased in runners after a three-day intensified exercise as a result of fatty acid oxidation [29]. On the other hand, Berton et al. [8] did not find any change in 3-hydroxybutyrate after a leg press resistance exercise. Our result might sugges<sup>t</sup> that this metabolic pathway is of negligible importance in trained standardbred horses performing acute exercise of short duration.

4-aminobutyrate ( γ-Aminobutiryc acid or GABA) is the most inhibitory neurotransmitter in the central nervous system [46] and its presence has been already demonstrated in human saliva [47] as well as in salivary glands of men and rats [48,49], being associated with its biosynthetic and metabolic enzymes. However, this is the first report showing the presence of GABA in equine saliva and the down-regulatory e ffect of exercise in this species. GABA and its receptors can be found in other non-neuronal organs but its role in peripheral tissue remains to be established, although it is known to be involved in cellular proliferation [48]. Authors suggested a role of GABA(A)-R in the suppression of salivary secretion in rat salivary glands [50]. Similarly, the down-regulation of salivary GABA during exercise in horses could be associated with this regulatory function of saliva secretion, possibly linked to thermoregulation.

Significantly lower levels of myo-inositol have been found both in the serum and saliva of horses after exercise. Myo-inositol promotes the maturation of pulmonary surfactants and supports respiratory function [51], modulates cytoskeleton dynamics, thus allowing alveolar cells to counteract collapsing forces and promoting mechanical stabilization of cell shape [52], and it recruits water and organic compounds in the alveolar space, decreasing surface tension through the formation of a biofilm layer at the interface [53]. Myo-inositol is also the most e ffective allosteric e ffector identified to date, being able to increase the tissue delivering of oxygen bound to hemoglobin [54]. Since its administration can improve sport performance in laboratory mice, its analogues have been suspected to be abused in the horse racing industry [55]. Up to now, no study about its e ffect in equine species has been performed. Recently, reduced concentrations of myo-inositol have been found in the bronchoalveolar lavage fluid of horses a ffected by equine asthma in comparison with healthy horses [56]. The exact role of this metabolite in physical exercise is not known, however, the decrease observed in both serum and saliva could indicate an impairment in the normal respiratory function during exercise.

#### **4. Materials and Methods**
