**2. Results**

#### *2.1. Urinary Metabolites Identification by Untargeted 1H-NMR*

A representative spectrum of the metabolites identified in the gira ffe's urine is reported in Figure 1. In this study, we identified 39 molecules (Table S1). These molecules mainly pertain to the classes of amino acids and derivatives and organic acids and derivatives. Hippurate (30.63%), creatinine (25.17%), and phenylacetylglycine (12.64%) were the most represented metabolites.

**Figure 1.** Portions of 1H-NMR spectra, representative of all the spectra obtained in this study. Each molecule's name appears over the NMR peak used for its quantification. To ease the visual inspection of each portion, a different spectrum with a convenient signal-to-noise ratio has been selected.

#### *2.2. E*ff*ects of Sampling Procedure and Location*

To check the potential influence of the different sampling methods, we wanted to collect pairs of samples during the same voiding, one directly and one from the ground. Unfortunately, we only succeeded in this task for one individual (Ronny). Among 39 quantified compounds, four molecules showed a variation of concentration higher than 50%, namely *p*-cresol sulfate, citrate, glycine, and benzoate. 1H-NMR signals for these compounds are reported in Figure 2. In detail, benzoate and glycine were more concentrated in the urine collected from the ground, while citrate and *p*-cresol sulfate showed the opposite trend. Overall, the 39 molecules showed a median difference between the two samples of 4.8%. As these observations were based only on one pair of samples from a single individual, we decided not to exclude these molecules from the subsequent analyses.

**Figure 2.** Representative sections of two spectra obtained from analyzing urine from the same giraffe (Ronny), collected directly (**blue line**) and from the ground (**red line**) during one urination, respectively.

To obtain hints about the potential effects of location on the metabolome of giraffe urine, we selected the samples from the locations BG (Parco Faunistico Le Cornelle) and FA (Zoosafari Fasanolandia), where most of the samples had been collected, and we set up a three-way ANOVA analysis aiming at excluding any effect related to gender or age. None of the molecules quantified appeared as significantly different in relation to zoo, so this variable was not considered in the subsequent analyses.

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

To obtain preliminary data on the effect of sex on the urinary metabolome, we focused on samples collected from adult, non-pregnan<sup>t</sup> individuals. Six molecules were found to be significantly (*p* < 0.05) affected by sex, as shown in Table 1.


**Table 1.** Metabolite concentrations (mmol/L, median (IQR)) in the adult group were significantly (*p* < 0.05) affected by sex, as assessed by *t*-test.

To have an overall view of the data, a robust principal component analysis (rPCA) model was calculated on their concentration, as shown in Figure 3.

**Figure 3.** rPCA model calculated on the concentration of the significantly different molecules between male and female giraffes. The scoreplot (**A**) represents with squares and circles females and males, respectively. The median of each sample group is represented by wide circles. The loading plot (**B**) reports the correlation between the importance of each substance over principal component 1 and its concentration. Gray bars highlight significant correlations (*p* < 0.05).

Three principal components (PCs) were accepted by the algorithm to depict the overall data features. PC 1, accounting for 59% of the variance thus represented, indeed significantly summarized the peculiarities connected to sex (*p* < 0.05), with female and male individuals appearing respectively at low and high PC scores. Among these molecules, hippurate, phenylacetylglycine, and thymine were more abundant in the urine of male individuals, while lactate, acetate, and succinate were more concentrated in the females' urine.

#### *2.4. E*ff*ect of Age on the Urinary Metabolome*

Age was found to significantly affect (*p* < 0.05) the concentration of three urinary metabolites, namely formate, alanine, and valerate, (Figure 4). To understand if their evolution was part of a trend spanning over the entire life of the giraffe, these molecules were used as a base for an rPCA model (Figure 5).

**Figure 4.** Boxplots showing the concentration of molecules significantly (*p* < 0.05) affected by age, as assessed by two-way ANOVA followed by Tukey post-hoc test.

**Figure 5.** rPCA model of the concentration of the molecules showing a significant difference among the giraffes grouped by age. The scoreplot (**A**) shows the samples from the three groups with squares (Young), circles (Adult), and triangles (Old). The median of each sample group is represented by wide circles. The boxplot (**B**) summarizes the positions of the samples along PC1 and compares them by two-way ANOVA, followed by Tukey post-hoc test. The loading plot (**C**) reports the correlation between the importance of each substance over PC 1 and its concentration. Gray bars highlight significant correlations (*p* < 0.05).

Three PCs were accepted by the algorithm to depict the overall data features. PC 1, accounting for 44.1% of the variance thus represented, summarized effectively the peculiarities connected to age (*p* < 0.05), with Young, Adult, and Old individuals appearing respectively at low, intermediate, and high PC scores. Among these molecules, formate and alanine were more abundant in young individuals, while valerate showed an opposite trend.

#### *2.5. Pregnancy Related Urinary Metabolome*

Urine samples were obtained from two female giraffes during and after pregnancy (Table S2). Despite the limited number of samples, it was possible to observe a variation of five metabolites during the pregnancy. These molecules showed consistent trends in the samples from both giraffes. All these molecules showed a relevant increase in concentration during the pregnancy, except for phenylacetylglycine, as shown in Table 2.


**Table 2.** Urinary metabolites (mmol/L) affected by pregnancy consistently across the two giraffes observed.

> 1 For readability, only molecules changing by more than 40% for both giraffes are shown.
