**3. Discussion**

The present paper describes one of the first studies ever devoted to the urinary metabolome of nonfarmed animals, and the very first focusing on the giraffe metabolome. Due to such paucity of studies on the topic, a key point that needs to be addressed before giraffe urine can be used for metabolomics studies is the possibility of relying on samples collected from the ground. Several aspects, in fact, make the collection of urine directly from the individual during urination highly impractical. To obtain a first insight on this point, we managed to collect the same urine sample either at the start of a spontaneous voiding or from the ground with a syringe at the end the voiding. The corresponding 1H-NMR spectra were highly superimposable, except for four molecules, namely benzoate, citrate, *p*-cresol sulfate, and glycine. The fact that the non-volatile glycine showed the greatest differences gave hints that the discrepancies could be mainly connected to dynamic variations in composition during urination, in agreemen<sup>t</sup> with Sink and Weinstein [16]. Modifications induced by the collection method could therefore be considered a confounding factor of lower entities than inhomogeneity in the composition of urine during voiding.

The 39 molecules identified give information about protein digestion, diet, gut-microbial co-metabolism, and energy production. Their quantitative observation therefore offers a handy perspective of the health status of giraffes, through a quintessentially non-invasive sampling method.

Comparisons with the urinary metabolome of other animals are also possible, giving indirect information about the differences in metabolism. An example of this possibility is offered by allantoin. This molecule is the fourth most concentrated in giraffe urine (Table S1), identically to yak (*Bos grunniens*) [17] and horse [18]. Differently from these strictly herbivorous animals, this molecule is the most concentrated in the urine of the giant panda [19], even if the giant panda consumes an amount of vegetables in relation to body weight (as much as 30%) much higher than ruminants or horses, which should lead to the lowest concentration of urinary allantoin [20]. This apparent contradiction leads to speculate that the main mechanism determining the concentration of allantoin in the urine of the above-mentioned animals is likely to be its renal reabsorption, which is very effective in strictly herbivorous animals [20].

#### *3.1. Sex A*ff*ects the Gira*ff*e Urine Molecular Profile*

In the current study acetate, succinate, and lactate concentrations appeared to be significantly higher in female giraffe urine, while hippurate, phenylacetylglycine, and thymine were more concentrated in male urine. For acetate, two of the authors of the present paper identified a similar situation in horse urine [18]. For the other molecules, indirect connections with published findings can be devised. There is an abundance of references, focusing on humans, showing that exercise leads to higher concentrations of acetate, succinate, and lactate in urine, and lower concentrations of thymine and hippurate [21–23]. Ginnett et al. showed that female giraffes spend more time walking, foraging, feeding, and traveling than males [24]. The two observations seem to sugges<sup>t</sup> that the sex-related differences observed in the urine of males and females may be partly due to the different daily activities. Contrary to the previously reported molecules, phenylacetylglycine is mainly a co-metabolite of gu<sup>t</sup> microorganisms, derived from valine, leucine, phenylalanine, lysine, or ornithine [25]. Its different concentration in relation to sex may therefore reflect peculiarities in gu<sup>t</sup> microbiota profiles or different foraging behaviors, similarly to what was recently observed in the giant panda [19]. Ginnett et al., in fact, demonstrated that males prefer larger bites than females, with potential consequences on the food, and in turn urine, metabolome profile [24]. It is tantalizing to speculate that the length of the neck, which is higher in males [5], may play a role too. In fact, Schüßler and Greven [26] found an allometric direct relationship between rumen-to-mouth distance and the duration of rumination intercycles, influencing in turn the digestive action of ruminal microorganisms.

#### *3.2. E*ff*ect of Age*

By removing the gender effect by two-way ANOVA, it was possible to focus on the effect of age. In parallel with previous studies in rats and humans [27,28], formate and alanine were negatively related to age. The trend observed for formate is very likely related to the gu<sup>t</sup> microbiome. In fact, in the gu<sup>t</sup> microbiota of the juvenile giraffes there is a prevalence of *Bacteroides* and *Acinetobacter* genera, responsible for the degradation of starch and cellulose to formate [29], while in the gu<sup>t</sup> of adult giraffes other genera tend to prevail, such as *Treponema* [30].

The concentration of amino acids in urine has been consistently linked to the turnover of muscle amino acids [18,31], with urinary concentration of alanine specifically related to exercise [32]. Therefore, the difference in the concentration of alanine could be ascribed to a variation of daily activity intensity along age.

#### *3.3. E*ff*ect of Pregnancy*

Early identification of pregnan<sup>t</sup> giraffes with maximum accuracy is an important issue for optimizing their management. Although some diagnostic methods (e.g., ultrasonography) have been described in domestic animals [33], their application to wild or captive animals is hindered by practical reasons. Metabolomics approaches seem in principle promising for setting up diagnostic methods that might be more convenient in specific contexts, due to the possibility to quantify a high number of molecules at the same time. However, previous studies performed in domestic animals were focused on serum [34,35], a sub-optimal sample from the point of view of non-invasivity. Therefore, despite the restricted number of samples analyzed in the present study, the obtained data can provide a preliminary urinary fingerprint of pregnancy in giraffes.

Taurine is an important amino acid during pregnancy and lactation, because it satisfies the needs of both the fetus and suckling infant. In our research, taurine excretion through urine increased during early pregnancy, consistent with human studies [36]. Taurine is rarely found in plants [37], so that herbivores cannot obtain a sufficient amount taurine from the diet. Remarkably, in ruminants the urinary taurine concentration is strongly diet-dependent, as can be inferred from the works of Bristow et al. on cows fed with maize silage compared to free grazing cows [38]. Diet is therefore likely to trigger biosynthetic pathways, such as the one leading to taurine from methionine [39]. Moreover, a specific pathway, converting homocysteine to taurine and glycine through cysteine, is known to become effective in early pregnancy [39]. This latter mechanism is a likely reason for the increasing trend of taurine excretion we found in the present work.

A further contribution to urine metabolome profile modifications may be due to changes in the gu<sup>t</sup> microbiota. In fact, among the molecules showing the greatest changes we found *p*-cresol sulfate and phenylacetylglycine, mainly described as gu<sup>t</sup> microorganism co-metabolites [11,25], absorbed at the intestinal level and then expelled through urine. Interestingly, the change in the concentration of both has been related, in humans, with alterations in the microbiota profile linked to inflammatory states [13,40], in which pregnancy is known to play a role [41]. Despite the very limited number of cases

here, these observations support the compelling possibility to use the urine metabolome to gain specific information about giraffe inflammatory status during pregnancy, as modulated by the gu<sup>t</sup> microbiota.

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

#### *4.1. Compliance with Ethical Requirements*

All the procedures related to animals respected the Directive 2010/63/EU of the European Parliament and of the Council of September 22, 2010 on the protection of animals used for scientific purposes (Article 1, Paragraph 1, Letter b) and the Italian legislation (D. Lgs. n. 26/2014, Article 2, Paragraph 1, Letter b).
