In the last years there has been an increasing awareness of the pivotal role played by buffalo as a valuable protein source particularly for tropical and subtropical countries, leading to a remarkable worldwide increase in buffalo population, currently estimated to sit at 207 million heads [1]. The buffalo is a short-day breeder, exhibiting improved reproductive performance during months characterized by decreasing day length [2,3]. Reproductive seasonality is right now the main constraint of buffalo farming, leading to discontinuity of milk supply during the year. In Italy, as natural calving pattern is opposite to milk market demand, the out of breeding mating strategy (OBMS) is used [3]. The OBMS undoubtedly allows a more even calving distribution throughout the year but, a higher incidence of embryonic mortality is recorded during the non-breeding season (NBS) [2,4,5]. The higher incidence of embryonic mortality is partly due to reduced luteal function, and hence decreased progesterone secretion [6], which is related to delayed embryo growth and is associated to transcriptomic and proteomic changes [7,8], resulting in impeding embryo attachment. However, embryonic mortality during the NBS in Italian Mediterranean buffalo is also caused by decreased oocyte developmental competence, as clearly indicated by lower cleavage and blastocyst rates after in vitro fertilization [9,10]. In Murrah buffalo heifers the lowest recorded oocyte quality during the NBS was associated to reduced intrafollicular levels of estradiol and IGF-1 [11]. The oocyte acquires developmental competence, i.e. the capability to undergo fertilization and embryo development, during the last phase of growth that is tightly coordinated with that of the follicle [12]. The acquisition of oocyte developmental competence is a gradual process involving a precisely modulated spatio-temporal expression of various genes [12,13]. It is known that microRNAs (MiRNAs) play an important role in regulating gene expression [14]. Interestingly, seasonal variations in miRNAs content and transcriptomic profile in both buffalo oocytes and follicular cells were recently demonstrated [15]. The seasonal dependent changes in gene expression likely result in different metabolite abundance, influencing indeed the metabolic profile of the follicle. Therefore, the knowledge of metabolic profile of the follicle is fundamental to unravel the causes of reduced oocyte competence during the NBS. Metabolites are the most reliable indicators of phenotypic traits, as intermediate and/or end products of metabolic pathways. Metabolomics allows a broad identification of low molecular weight metabolites, that are the downstream products of genome, transcriptome and proteome expression [16,17] present in biological fluids, cells and tissues, and can provide a picture of the dynamic variations in response to environmental or genetic factors [18]. Currently several techniques are available for metabolomics, such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), Fourier transform infrared spectroscopy, Raman spectroscopy and nuclear magnetic resonance spectroscopy [17], which has been considered one of the strongest techniques for biological fluid research [19]. The follicular microenvironment where the oocyte grows certainly influences oocyte competence. It is known that follicular fluid composition, influenced by climate [20] and nutrition [21] affects the oocyte maturation process [22]. Metabolomic analysis of the follicular fluid has been carried out in cattle allowing the identification of some predictive markers for oocyte developmental competence [23]. Furthermore, variations in total fatty acid and amino acid profile were recorded in follicular fluid from cows compared with heifers, with low and high fertility, respectively [24]. To the best of our knowledge, metabolomics has so far not been applied to buffalo female reproduction. Therefore, the purpose of this experiment was to evaluate whether the season influences the metabolite content and metabolic pathways within the ovarian follicle in this species, to unravel the causes of the reduced competence during NBS and lay the basis for further studies to develop corrective strategies. To do so, we investigated the metabolomic profile of various components of the buffalo ovarian follicle in relation to season, such as follicular fluid, follicular cells, cumulus cells and oocytes by H1 Nuclear Magnetic Resonance (NMR) spectroscopy.