**1. Introduction**

According to the International Union for Conservation of Nature (IUCN), gira ffe (*Gira*ff*a camelopardalis*) is declared a vulnerable species [1]. Moreover, di fferent measures have been taken to monitor and protect gira ffe population. For example, the International Union for Conservation of Nature (IUCN) Species Survival Commission (SSC) Gira ffe and Okapi Specialist Group (GOSG) was established with the aim of studying and guaranteeing the conservation needs of this species (https://www.gira ffidsg.org/). In addition, from November 26, 2019, gira ffes are included in Appendix II of the CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) to improve its protection, subjecting it to strict regulation (https://www.cites.org/).

Zoos represent a significant part of the protection strategy for gira ffes, with projects explicitly aimed at protecting endangered species and pursuing high standards of animal welfare [2]. In these structures, however, gira ffes may be subjected to sources of stress that reverberate negatively on individual and social behaviors [3]. Causes of stress could be represented by the presence of visitors and attendants [4]. Among the e fforts that have been made to reduce the stressors, some are devoted to developing protocols to evaluate their general health status that do not involve immobilization, but are based on indirect methods [3]. In fact, gira ffes are particularly prone to anesthetic-related complications and death, due to their unique cardiovascular system, making immobilization a risky practice [5,6].

The possibility of obtaining information from urine collected from the ground seems particularly attractive from this point of view, but the literature on this type of sampling is absent for gira ffes and it has been only reported in okapi [7]. Indeed gira ffes have been studied more for their iconic height and the mechanisms existing at the cardiovascular level to counterbalance the consequent state of primary hypertension [8–10].

Among the completely unexplored characteristics of gira ffe urine is its metabolome, the ensemble of low weight molecules produced by the cellular metabolism. Studies carried out by liquid chromatography–mass spectrometry (LC/MS) or by proton nuclear magnetic resonance spectroscopy ( 1H-NMR) on humans and other animals sugges<sup>t</sup> that the gira ffe's urinary metabolome may be particularly informative about the general health of the animal. In horse urine, molecules revealing the action of the intestinal microbiota were found in micromolar concentrations [11,12]. Molecular patterns of the urinary metabolome linked to inflammatory processes have been identified in humans [13]. Urinary profile responses to the calorie content of the diet were identified in rat [14]. The e ffects of heat stress were studied in cattle by metabolomic profiling of urine [15]. Indeed, the use of urine as a source of biological data in gira ffes could be a suitable alternative, due to its non-invasive approach that could avoid the immobilization of animals.

Among the analytical platforms capable of fulfilling the requirements, proton nuclear magnetic resonance spectroscopy (1H-NMR) has been widely used for the investigation of urine metabolomes, taking advantage of its high reproducibility and minimal sample preparation.

In the present study, we wanted to verify the feasibility of 1H-NMR based metabolomic studies focusing on the urine of gira ffes. For this purpose, we characterized the molecular profile of healthy gira ffes held in captivity to obtain preliminary quantitative values that could be applied for the diagnosis of diseases a ffecting this animal. Moreover, the samples collected gave the opportunity to have a first insight about the influence of important physiological factors, such as the sex and age of the subjects, on the urinary metabolomic profile.
