H OH metabolites OH Article First Insights into the Urinary Metabolome of Captive Giraffes by Proton Nuclear Magnetic Resonance Spectroscopy 1, 2, 2 1, Chenglin Zhu y , Sabrina Fasoli y, Gloria Isani and Luca Laghi * 1 Department of Agro-Food Science and Technology, University of Bologna, 47521 Cesena, Italy; [email protected] 2 Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia, 40064 Bologna, Italy; [email protected] (S.F.); [email protected] (G.I.) * Correspondence: [email protected]; Tel.: +39-0547-338105 These authors contributed equally to this work. y Received: 13 March 2020; Accepted: 15 April 2020; Published: 17 April 2020 Abstract: The urine from 35 giraffes was studied by untargeted 1H-NMR, with the purpose of obtaining, for the first time, a fingerprint of its metabolome. The metabolome, as downstream of the transcriptome and proteome, has been considered as the most representative approach to monitor the relationships between animal physiological features and environment. Thirty-nine molecules were unambiguously quantified, able to give information about diet, proteins digestion, energy generation, and gut-microbial co-metabolism. The samples collected allowed study of the effects of age and sex on the giraffe urinary metabolome. In addition, preliminary information about how sampling procedure and pregnancy could affect a giraffe’s urinary metabolome was obtained. Such work could trigger the setting up of methods to non-invasively study the health status of giraffes, which is utterly needed, considering that anesthetic-related complications make their immobilization a very risky practice. Keywords: captive giraffes; urine; metabolomics; 1H-NMR 1. Introduction According to the International Union for Conservation of Nature (IUCN), giraffe (Giraffa camelopardalis) is declared a vulnerable species [1]. Moreover, different measures have been taken to monitor and protect giraffe population. For example, the International Union for Conservation of Nature (IUCN) Species Survival Commission (SSC) Giraffe and Okapi Specialist Group (GOSG) was established with the aim of studying and guaranteeing the conservation needs of this species (https://www.giraffidsg.org/). In addition, from November 26, 2019, giraffes 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 giraffes, with projects explicitly aimed at protecting endangered species and pursuing high standards of animal welfare [2]. In these structures, however, giraffes 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 efforts 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, giraffes are particularly prone to anesthetic-related complications and death, due to their unique cardiovascular system, making immobilization a risky practice [5,6]. Metabolites 2020, 10, 157; doi:10.3390/metabo10040157 www.mdpi.com/journal/metabolites Metabolites 2020, 10, x FOR PEER REVIEW 2 of 13 Metabolites 2020, 10, 157 2 of 13 complications and death, due to their unique cardiovascular system, making immobilization a risky practice [5,6]. The possibilitypossibility of of obtaining obtaining information information from urinefrom collectedurine collected from the from ground the seems ground particularly seems attractiveparticularly from attractive this point from of view,this point but theof view, literature but the on thisliterature type of on sampling this type is of absent sampling for gira is absentffes and for it hasgiraffes been and only it reportedhas been in only okapi reported [7]. Indeed in okapi gira ff[7].es haveIndeed been giraffes studied have more been for studied their iconic more height for their and theiconic mechanisms height and existing the mechanisms at the cardiovascular existing level at the to counterbalance cardiovascular the level consequent to counterbalance state of primary the hypertensionconsequent state [8– 10of]. primary hypertension [8–10]. Among thethe completely completely unexplored unexplored characteristics characteristics of gira offf egiraffe urine isurine its metabolome, is its metabolome, the ensemble the ofensemble low weight of low molecules weight molecules produced produced by the cellular by the metabolism.cellular metabolism. Studies Studies carried carried out by out liquid by chromatography–massliquid chromatography–mass spectrometry spectrometry (LC/MS) (LC/MS or by proton) or nuclearby proton magnetic nuclear resonance magnetic spectroscopy resonance (spectroscopy1H-NMR) on (1 humansH-NMR) andon humans other animals and other suggest animals that suggest the gira thatffe’s the urinarygiraffe's metabolomeurinary metabolome may be particularlymay be particularly informative informative about the about general the health general of the health animal. of the In horse animal. urine, In moleculeshorse urine, revealing molecules the actionrevealing of the the intestinal action of microbiota the intestinal were microbiota found in micromolar were found concentrations in micromolar [11 ,12concentrations]. Molecular patterns[11,12]. ofMolecular the urinary patterns metabolome of the linkedurinary to inflammatorymetabolome linked processes to haveinflammatory been identified processes in humans have been [13]. Urinaryidentified profile in humans responses [13]. to Urinary the calorie profile content responses of the dietto the were calorie identified content in of rat the [14 diet]. The were effects identified of heat stressin rat were[14]. studiedThe effects in cattle of heat by metabolomicstress were studie profilingd in of cattle urine by [15 metabolomic]. Indeed, the profiling use of urine of asurine a source [15]. ofIndeed, biological the use data of in urine giraff ases a could source be of a suitable biological alternative, data in giraffes due to itscould non-invasive be a suitable approach alternative, that could due avoidto its non-invasive the immobilization approach ofanimals. that could avoid the immobilization of animals. Among the analytical platforms capable of fulfilling fulfilling the requirements, proton nuclear magnetic resonance spectroscopy (1H-NMR) has been widely used used for for the the investigation investigation of of urine urine metabolomes, metabolomes, taking advantage of its high reproducibility andand minimalminimal samplesample preparation.preparation. In the present study, wewe wantedwanted toto verifyverify thethe feasibilityfeasibility ofof 11H-NMR based metabolomic studies focusing on the urine of giragiraffes.ffes. For this purpose, we characterized the molecular profile profile of healthy giragiraffesffes held in captivity to obtain preliminary quantitative values that could be applied for the diagnosis of diseases aaffectingffecting this animal. Moreover, the samples collected gave the opportunity to have a firstfirst insight about the influenceinfluence of important physiological factors, such as the sex and age of the subjects, on the urinary metabolomic profile.profile. 2. Results 1 2.1. Urinary Metabolites Id Identificationentification by Untargeted 1H-NMR A representativerepresentative spectrum spectrum of of the the metabolites metabolites identified identified in the in gira theff e’sgiraffe’s urine isurine reported is reported in Figure in1. InFigure this 1. study, In this we study, identified we identified 39 molecules 39 molecules (Table S1). (Table These S1). molecules These molecules mainly pertainmainly pertain to the classes to the ofclasses amino of acidsamino and acids derivatives and derivatives and organic and acids organic and acids derivatives. and derivatives. Hippurate Hippurate (30.63%), creatinine(30.63%), (25.17%),creatinine and (25.17%), phenylacetylglycine and phenylacetylglycine (12.64%) were (12.64%) the most were represented the most represented metabolites. metabolites. Figure 1. PortionsPortions of of1H-NMR1H-NMR spectra, spectra, representative representative of all of the all spectra the spectra obtained obtained in this in study. thisstudy. Each Eachmolecule’s molecule’s name nameappears appears over overthe theNMR NMR peak peak used used for for its its quantification. quantification. To To ease ease the the visual inspection of each portion, a different spectrum with a convenient signal-to-noise ratio has been selected. Metabolites 2020, 10, x FOR PEER REVIEW 3 of 13 Metabolitesinspection2020, 10 of, 157 each portion, a different spectrum with a convenient signal-to-noise ratio has been3 of 13 selected. 2.2. Effects Effects of Sampling Procedure and Location To checkcheck thethe potentialpotential influenceinfluence of thethe didifferenfferentt samplingsampling methods,methods, we wantedwanted 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 quantified compounds,compounds, four molecules showed a variation of concentrationconcentration higher than 50%, namelynamely p-cresol-cresol sulfate,sulfate, citrate,citrate, glycine,glycine, and 1 benzoate. 1H-NMRH-NMR signals signals for for these these compounds compounds