Research in Veterinary Science 153 (2022) 127–136 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc Metabolic profiling of preovulatory follicular fluid in jennies Jaime Catalán a, b, c, d, Iris Martínez-Rodero a, Iván Yánez-Ortiz a, b, c, Yentel Mateo-Otero b, c, Ana Flores Bragulat e, Pau Nolis f, Augusto Carluccio d, Marc Yeste b, c, g, 1, *, Jordi Miró a, 1, * a Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, ES-08193 Cerdanyola del Vallès, Barcelona, Spain b Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, ES-17003 Girona, Spain c Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, ES-17003 Girona, Spain d Faculty of Veterinary Medicine, University of Teramo, Loc. Piano d'Accio, IT-64100 Teramo, Italy e Equine Production Laboratory, Faculty of Agronomy and Veterinary Medicine, National University of Río Cuarto, AR- X5800 Río Cuarto, Córdoba, Argentina f Nuclear Magnetic Resonance Facility, Autonomous University of Barcelona, Bellaterra, ES-08193 Cerdanyola del Vallès, Spain g Catalan Institution for Research and Advanced Studies (ICREA), ES-08010 Barcelona, Spain. A R T I C L E I N F O A B S T R A C T Keywords: Follicular fluid is formed from the transudation of theca and granulosa cells in the growing follicular antrum. Its Donkey main function is to provide an optimal intrafollicular microenvironment to modulate oocyte maturation. The aim Equids of this study was to determine the metabolomic profile of preovulatory follicular fluid (PFF) in jennies. For this Ovary purpose, PFF was collected from 10 follicles of five jennies in heat. Then, PFF samples were analysed by nuclear Preovulatory follicle Follicular fluid magnetic resonance (NMR) and heteronuclear single quantum correlation (2D 1H/13C HSQC). Our study revealed Metabolomic the presence of at least 27 metabolites in the PFF of jennies (including common amino acids, carboxylic acids, Metabolites amino acid derivatives, alcohols, saccharides, fatty acids, and lactams): 3-hydroxybutyrate, acetate, alanine, betaine, citrate, creatine, creatine phosphate, creatinine, ethanol, formate, glucose, glutamine, glycerol, glycine, hippurate, isoleucine, lactate, leucine, lysine, methanol, phenylalanine, proline, pyruvate, threonine, tyrosine, valine, and τ-methylhistidine. The metabolites found here have an important role in the oocyte development and maturation, since the PFF surrounds the follicle and provides it with the needed nutrients. Our results indicate a unique metabolic profile of the jennies PFF, as it differs from those previously observed in the PFF of the mare, a phylogenetically close species that is taken as a reference for establishing reproductive biotechnology techniques in donkeys. The metabolites found here also differ from those described in the TCM-199 medium enriched with fetal bovine serum (FBS), which is the most used medium for in vitro oocyte maturation in equids. These dif­ ferences would suggest that the established conditions for in vitro maturation used so far may not be suitable for donkeys. By providing the metabolic composition of jenny PFF, this study could help understand the physiology of oocyte maturation as a first step to establish in vitro reproductive techniques in this species. 1. Introduction metabolomics represents the ultimate answer of an organism to envi­ ronmental influences, genetic alterations, or disease (Dettmer and Metabolomics is a modern field in analytical biochemistry that can Hammock, 2004). be understood as the final step in the “omics” cascade (Dettmer and Metabolomics examines small molecules (<1000 Da), the metabo­ Hammock, 2004; Jurowski et al., 2017). Whereas genomics investigates lites, which are the end products of cellular processes that are affected the complete genome to decipher gene function, transcriptomics gives a by the physiological state of a cell at any given time (Dumesic et al., more functional focus by measuring only the expressed genes and pro­ 2015). In the case of ovarian cell types, their metabolites are found in a teomics tries to comprehend the role of translated proteins, complex dynamic biological fluid that surrounds the developing oocyte: * Corresponding authors at: Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, ES-17003 Girona, Spain; Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, ES-08193 Cerdanyola del Vallès, Barcelona, Spain E-mail addresses: marc.yeste@udg.edu (M. Yeste), jordi.miro@uab.cat (J. Miró). 1 Joint senior authors. https://doi.org/10.1016/j.rvsc.2022.10.026 Received 2 August 2022; Received in revised form 21 September 2022; Accepted 26 October 2022 Available online 3 November 2022 0034-5288/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). J. Catalán et al. Research in Veterinary Science 153 (2022) 127–136 the so-called follicular fluid (FF) (Singh and Sinclair, 2007; Nel-Themaat attempts to use FF for the in vitro maturation (IVM) of oocytes in and Nagy, 2011; Mariani and Bellver, 2018). The FF is formed from the different species have been made (Douet et al., 2017; Zhao et al., 2020; transudation of theca and granulosa cells in the growing follicular Horikawa et al., 2019; Cruz et al., 2014; Somfai et al., 2012; Azari- antrum (Guerreiro et al., 2018) and contains a variety of molecules, such Dolatabad et al., 2021; Tesarik and Mendoza, 1995; Tesarik and Men­ as steroid hormones, polysaccharides, proteins, reactive oxygen species doza, 1997). It has been demonstrated that supplementing the media (ROS), antioxidants, and metabolites (Mariani and Bellver, 2018). The used for IVM with FF resulted in improved oocyte quality and matura­ function of these components is to provide an optimal intrafollicular tion rates (Zhao et al., 2020; Horikawa et al., 2019; Azari-Dolatabad microenvironment to modulate oocyte and follicular maturation (Bian­ et al., 2021), sperm penetration (Somfai et al., 2012), and subsequent chi et al., 2016), facilitate essential communication between somatic embryonic development (Horikawa et al., 2019; Cruz et al., 2014; Azari- and germ cells to acquire oocyte competence (Mariani and Bellver, Dolatabad et al., 2021). In bovine, Azari-Dolatabad et al. (Azari-Dola­ 2018), protect follicular cells from oxidative damage (Ambekar et al., tabad et al., 2021) observed that these beneficial effects rely upon the 2013; Freitas et al., 2017; Gupta et al., 2011) and contribute to the amount of FF added to the IVM medium, the composition of that me­ oocyte extrusion during ovulation (Richards et al., 1998). The compo­ dium (serum vs. serum-free), and the type of culture system (group vs. sition and quantity of FF within the follicle changes during the different individual). In horses, supplementing the IVM medium with FF also has follicular developmental stages, from initial growth to atresia (Freitas a positive effect (Spacek and Carnevale, 2018; Dell'Aquila et al., 1997; et al., 2017; Gupta et al., 2011; Yang et al., 2020; Wise, 1987; Fortune Caillaud et al., 2008; Douet et al., 2017); however, whether this is also et al., 2004), whereas other factors associated to FF variations are ani­ the case of the donkey - a species that, despite being phylogenetically mal age (Takeo et al., 2017; Bender et al., 2010), follicular size and day close to the horse, has a different reproductive strategy, remains of oestrous (Gupta et al., 2011; Wise, 1987). unaddressed. Because the FF constitutes the immediate microenvironment of the Most wild equids are currently endangered or threatened, while oocyte, it provides the nutrients, growth factors and hormones necessary many breeds of domestic horses and donkeys are in risk of extinction. to promote its growth and maintain its viability (Dumesic et al., 2015). This situation led to the development of various conservation programs Thus, the composition of FF significantly affects follicular growth and to focus on the need of storing genomic resources (Douet et al., (Freitas et al., 2017; Gupta et al., 2011), the quality and quantity of 2017). This, added to the growing demand for new uses (milk produc­ matured oocytes (Dumesic et al., 2015; Guerreiro et al., 2018; Takeo tion, cosmetics, forestry, rural tourism, leisure…), promotes an et al., 2017), in vitro fertilization rate (Nagy et al., 2019; Botero-Ruiz increasing interest in developing studies to understand and improve et al., 1984) and pregnancy outcome after embryo transfer (Boakari donkey reproductive performance (Martini et al., 2018; Miró et al., et al., 2021) by accumulating products from all types of metabolism 2020). (Yang et al., 2020). In fact, multiple FF components have been identified Despite advances in donkey sperm cryopreservation, the use of as biomarkers of oocyte developmental competence (Kreiner et al., frozen-thaw semen for artificial insemination is limited by the low 1987; Revelli et al., 2009; O'Gorman et al., 2013) and in vitro embryo pregnancy rates obtained in jennies (Miró et al., 2020; Álvarez et al., development (Kafi et al., 2017; Sinclair et al., 2008). 2019; Vidament et al., 2009; Canisso et al., 2011; Oliveira et al., 2012). In equids, the production of in vivo-derived embryos from donors is Thus, the development of other reproductive biotechnologies such as in limited by the lack of efficiency in superovulation treatments (Squires vitro production (IVP) of embryos could be important for breeding in this et al., 2003; Smits et al., 2012; Meyers-Brown et al., 2011), thus species. In addition, IVP of embryos and their cryopreservation would reducing the number of embryos available to one per oestrus cycle allow the preservation of the male and female genetics, being a fast (Smits et al., 2012; Goudet et al., 2015; Deleuze et al., 2018). In addi­ method to restore a breed (Douet et al., 2017; Woelders et al., 2012). tion, the large size of recovered in vivo embryos makes them “bad can­ Although the first experiments of oocyte IVM (Goudet et al., 2015; didates” for cryopreservation (Choi and Hinrichs, 2017; Herrera et al., Deleuze et al., 2018; Zhao et al., 2011; Abdoon et al., 2014), in vitro 2015). In vivo-derived embryos are normally recovered after 7–8 days fertilization (IVF) (Goudet et al., 2015; Deleuze et al., 2018) and even from ovulation in the blastocyst stage and have a diameter of >300 μm, vitrification (Douet et al., 2019) were performed in jennies, maturation due to a blastocoelic cavity full of fluid that makes more difficult the and sperm penetration rates are still very low and no IVP embryos have exchange of water by cryoprotectants (Squires et al., 2003; Deleuze been obtained yet. et al., 2018; Choi and Hinrichs, 2017; Herrera et al., 2015; Hinrichs and For the above reasons, further knowledge on the metabolic profile of Hinrichs, 2012). For this reason, the very first step for IVP of embryos donkey follicular fluid is important for the development of reproductive from donors is widely documented in horses: the ultrasound-guided biotechnologies such as IVM, IVF and IVP of embryos in this species. As oocyte retrieval, or Ovum Pick Up (OPU) (Kanitz et al., 1995; Purcell far as we are concerned, there are no previous reports on the composi­ et al., 2007; Jacobson et al., 2010; Galli et al., 2007; Spacek and Car­ tion of jenny FF. Thus, the objective of this study was to describe, for the nevale, 2018; Dell'Aquila et al., 1997; Caillaud et al., 2008; Douet et al., first time, the metabolic profile of preovulatory follicular fluid (PFF) 2017). After collection and IVM, oocytes are then fertilized by intra­ collected from Catalonian jennies, as a preliminary step to set up better cytoplasmic sperm injection (ICSI), which in horses is very efficient and conditions for IVM in this species. the only option, as, to date, conventional IVF does not work in this species (Choi and Hinrichs, 2017; Herrera et al., 2015; Jacobson et al., 2. Materials and methods 2010; Galli et al., 2007; Spacek and Carnevale, 2018; Dell'Aquila et al., 1997; Caillaud et al., 2008; Douet et al., 2017; Leemans et al., 2016). 2.1. Chemicals and suppliers OPU and ICSI are, therefore, established procedures that have been widely implemented to produce horse embryos in vitro, both for scien­ Unless otherwise stated, all chemicals and reagents were purchased tific purposes and breeding from infertile and sporting mares (Galli from Merck (Merck KgaA, Darmstadt, Germany). et al., 2014). While, as it is one of the key steps for embryo IVP, much research has been conducted regarding the IVM of horse oocytes 2.2. Animals and samples (Jacobson et al., 2010; Galli et al., 2007; Spacek and Carnevale, 2018; Dell'Aquila et al., 1997; Caillaud et al., 2008; Fernández-hernández FF samples were obtained from 10 preovulatory follicles of five et al., 2020; Lewis et al., 2020a; Lewis et al., 2020b; Walter et al., 2019; Catalonian jennies (two follicles per jenny) between 3 and 12 years old. Ascari et al., 2017; Pereira et al., 2019), little has been made in the case Animals were kept in paddocks, fed with grain forage, straw and hay, of donkeys (Zhao et al., 2011). provided with water ad libitum, and housed at the Equine Reproduction Based on its properties and role in physiological conditions, several Service, Autonomous University of Barcelona (Bellaterra, Cerdanyola 128 J. Catalán et al. Research in Veterinary Science 153 (2022) 127–136 del Vallès, Spain), which works under strict health control and animal 8.4.0, GraphPad Software LLC; San Diego, CA, USA) was used to design welfare protocols. This Service already works under the approval of the the graphics. Concentrations of each metabolite identified in jenny PFF Generalitat de Catalunya (Spain), and the study was approved by the were analysed by descriptive statistics, where the mean, SEM, and range Ethics Committee of the Autonomous University of Barcelona (Code: were established. CEEAH 1424). The reproductive tract of jennies was carefully examined by ultra­ 3. Results sound prior to the extraction of PFF. Samples were obtained from fol­ licles ≥35 mm in diameter accompanied by uterine edema and without 3.1. Metabolites identified in jenny PFF corpus luteum in the ovaries, which was considered preovulatory ac­ cording to the described by Taberner et al. (Taberner et al., 2008) for The rationale behind studying the PFF metabolome is supported by this breed. By echo-guided flank puncture using a 20-mL plastic syringe the crucial function of this fluid, as it supports oocyte maturation, attached to a 20-g hypodermic needle and ovary displacement to the ovulation and fertility, and also plays a role in embryo development and flank by transrectal palpation, approximately 15 mL of PFF per follicle implantation (Singh and Sinclair, 2007; Guerreiro et al., 2018; Fortune were aspirated. Then, the total volume of PFF was split into 1-mL ali­ et al., 2004; Revelli et al., 2009). quots and kept in Eppendorf tubes at − 80 ◦ C until further use. The present work investigated for the first time the metabolic profile of jennies' PFF using 1H NMR, followed by a heteronuclear simple 2.3. Preparation of PFF samples for NMR quantum correlation for metabolite identification purposes. Our study revealed the presence of at least 27 metabolites (see Table 1), including An aliquot of each PFF sample was thawed on ice, vortexed and common amino acids, carboxylic acids, amino acid derivatives, alcohols, centrifuged through 0.5-mL Amicon® ultracentrifuge filters at 14,000 saccharides, fatty acids, and lactams. A correlation heat map (see Sup­ ×g and 4 ◦ C for 90 min to remove protein and cell debris. These filters plementary Fig. 1) revealed that most metabolites correlated positively had been previously washed four times by adding 500 μL PBS and with each other across the 10 PFF samples, except for ethanol with centrifuging at 14,000 ×g and 4 ◦ C for 20 min. methanol, lactate and glycerol, which presented a negative correlation. After centrifugation, 200 μL of each eluted PFF sample were diluted Representative 1H NMR spectra with expanded areas for metabolite with 100 μL PBS (containing 10% D2O with 0.33% DSS; pH 7.4) and 300 assignment acquired at 600.13 MHz is shown in Supplementary Fig. 2. μL D2O. Samples were then transferred to 5-mm Wilmad® NMR tubes Expanded area of 1H/13C HSQC NMR and metabolite assignment of a for NMR profile acquisition. representative sample acquired at 500 MHz equipped with a cryprobe is available in Supplementary Fig. 3. 2.4. NMR spectra 3.2. Comparison of jenny preovulatory follicular fluid metabolites with To obtain 1H NMR spectra, a Bruker 600-MHz AVANCE III NMR those found in the mare and in TCM-199 medium spectrometer (Bruker Biospin, Rheinstetten, Germany), operating at a 1 H frequency of 600.13 MHz and 300 K with a previous equilibration Table 2 shows the comparison of metabolites described in mare PFF time (10 min), was used. The 1D-1H-nuclear Overhauser effect spec­ by Fernández-Hernández et al. (Fernández-hernández et al., 2020) and troscopy (1D-NOESY) pulse sequence from the Bruker library was used those included in the composition of commercial TCM-199 medium with (noesygppr1d). The parameters applied were: (1) mixing time: 100 ms those found in our study in jenny. Of the 27 metabolites identified in (d8); (2) recovery delay: 2 s (d1); (3) 90◦ pulse: 10.42 μs (p1); (4) jenny PFF, 12 coincided with those described in the mare. These me­ spectral width: 7211.539 Hz; (5) spectral size: 32 k; (6) number of scans: tabolites were acetate, alanine, citrate, creatine, creatine phosphate, 256; and (7) acquisition time: 2.27 s. glucose, glycine, isoleucine, lactate, leucine, threonine, and valine. For metabolite identification, a 2D 1H/13C HSQC (Heteronuclear Concentrations of acetate, citrate and glucose did not differ greatly from Single Quantum Correlation) was acquired in a representative sample. A the mean reported in the mare. For the rest of metabolites, there were Bruker 500-MHz AVANCE-III NMR spectrometer equipped with a triple marked differences of a lower concentration in the PFF of the donkey channel cryoprobe and z-gradient was used. The experiment was run than in the mare, except for valine, which has a much higher concen­ using a slight modification of the original pulse sequence hsqcf3gpph19 tration in the donkey. from Bruker library; this modification consisted of adding water pre­ On the other hand, eight of the 27 metabolites identified in jenny PFF saturation during the recovery delay and changing the original f3 coincided with those included in the composition of TCM-199. These channel (usually used for 15N) to f2 channel for 13C. Relevant acquisition metabolites were acetate, alanine, glucose, glycine, isoleucine, leucine, parameters were (1) recovery delay d1 = 1 s; (2) constant for JCH threonine and valine. While the concentrations of alanine and glucose in evolution = 140 Hz; (3) Spectral size = 2 K × 128; (4) number of scans = jenny PFF did not differ greatly from those in TCM-199, those of the 400; (5) Spectral widths = 15.6 ppm for 1H and 100 ppm for 13C. remaining metabolites, except for acetate and valine, are considerably See Supplementary Material for 1H and HSQC spectral images and lower in jenny PFF than in TCM-199. metabolite assignment. 4. Discussion 2.5. Data processing and analysis In equids, the production of in vivo-derived embryos from donors is Spectra were processed and analysed through the Chenomx 8.0 limited by the lack of efficiency in superovulation treatments (Squires profiler software (Chenomx Inc., Edmonton, Canada). This software et al., 2003; Smits et al., 2012; Meyers-Brown et al., 2011), thus contains tools for automatic phase, baseline correction, reference cali­ reducing the number of embryos available to one per oestrus cycle bration and a library of metabolites for spectral profiling. The concen­ (Smits et al., 2012; Goudet et al., 2015; Deleuze et al., 2018). In addi­ tration of each metabolite identified in SP was calculated based on DSS tion, the large size of recovered in vivo embryos makes them “bad can­ concentration (0.216 mmol/L). didates” for cryopreservation (Choi and Hinrichs, 2017; Herrera et al., 2015). In vivo-derived embryos are normally recovered after 7–8 days 2.6. Statistical analyses from ovulation in the blastocyst stage and have a diameter of >300 μm, due to a blastocoelic cavity full of fluid that makes more difficult the The statistical package R (V 4.0.3, R Core Team; Vienna, Austria) was exchange of water by cryoprotectants (Squires et al., 2003; Deleuze used to run statistical analyses, whereas the GraphPad Prism software (V et al., 2018; Choi and Hinrichs, 2017; Herrera et al., 2015; Hinrichs and 129 J. Catalán et al. Table 1 Mean ± SEM and range of concentrations, chemical nature, KEGG pathways, cell localisation and identification (PubChem/KEGG database) of metabolites in jenny preovulatory follicular fluid (PFF) samples (n = 10). Metabolite Mean (mM) ± SEM (mM) Range (mM) Chemical nature1 KEGG pathways1 Cellular localisation2 PubChem CID2/KEGG entry1 3-Hydroxybutyrate 0.414 ± 0.048 0.242–0.655 Hydroxy fatty acid Intermediate metabolite – 3,541,112 Regulatory signaling - Acetate 1.333 ± 0.183 0.707–2.234 Organic monocarboxylic acid Glycolysis/Gluconeogenesis – 175 Pyruvate metabolism C00033 Alanine 0.355 ± 0.044 0.204–0.593 Common amino acid Biosynthesis of amino acids Cytoplasm 602 Protein digestion and absorption Lysosome C00041 Mitochondria Peroxisome Extracellular Betaine 0.106 ± 0.019 0.038–0.213 Amino acid derivative Glycine, serine and threonine metabolism – 247 ABC transporters C00719 Citrate 0.343 ± 0.034 0.225–0.544 Tricarboxylic acid Citrate cycle (TCA cycle, Krebs cycle) – 31,348 Reductive citrate cycle (Arnon-Buchanan cycle) C00158 Creatine 0.111 ± 0.010 0.061–0.175 Composed amino acid Metabolic pathways (ATP) Cytoplasm 586 Biosynthesis of amino acids2 Mitochondria C00300 Extracellular Creatine phosphate 0.089 ± 0.012 0.050–0.168 Phosphoamino acid Rapid source of energy (ADP➔ATP)2 Mitochondria 9,548,602 C02305 Creatinine 0.063 ± 0.010 0.029–0.129 Lactam Creatine pathway Cytoplasm 588 C00791 Ethanol 0.794 ± 0.144 0.467–2.033 Primary alcohol Glycolysis / Gluconeogenesis Cytoplasm 702 Pyruvate metabolism Peroxisome C00469 Inflammatory mediator regulation of TRP channels Extracellular Formate 0.815 ± 0.088 0.453–1.188 Organic monocarboxylic acid Pyruvate metabolism – 283 Degradation of aromatic compounds C00058 130 Glucose 4.820 ± 0.424 2.954–6.612 Monosaccharide Glycolysis / Gluconeogenesis – 5793 Starch and Sucrose metabolism C00031 Amino sugar and Nucleotide sugar metabolism Glutamine 0.465 ± 0.050 0.245–0.735 Common amino acid Nucleotide metabolism Extracellular 5961 ABC transporters / GABAergic synapse Mitochondria C00303 Protein digestion and absorption Glycerol 0.915 ± 0.129 0.377–1.756 Sugar alcohol Regulation of lipolysis Extracellular 753 Thermogenesis Mitochondria C00116 Metabolic pathways Glycine 0.436 ± 0.053 0.257–0.694 Common amino acid Protein digestion and absorption Extracellular 750 ABC transporters Lysosome C00037 Glutathione metabolism Mitochondria Biosynthesis of amino acids and cofactors Peroxisome Hippurate 0.053 ± 0.007 0.020–0.081 Carboxylic acid Metabolic pathways Cytoplasm 464 C01586 Research in Veterinary Science 153 (2022) 127–136 Isoleucine 0.139 ± 0.012 0.089–0.199 Common amino acid Protein digestion and absorption Cytoplasm 6303 ABC transporters Extracellular C00407 Biosynthesis of amino acids Mitochondria Lactate 2.091 ± 0.345 1.306–4.270 Hydroxycarboxylic acid Glycolysis / Gluconeogenesis – 91,435 cAMP signaling pathway C00186 Glucagon signaling pathway Leucine 0.179 ± 0.018 0.101–0.248 Common amino acid 2-Oxocarboxylic acid metabolism Extracellular 6106 Biosynthesis of amino acids Mitochondria C00123 ABC transporters Protein digestion and absorption Lysine 0.131 ± 0.013 0.076–0.193 Common amino acid 2-Oxocarboxylic acid metabolism Cytoplasm 5962 Biosynthesis of amino acids Extracellular C00047 ABC transporters Mitochondria Protein digestion and absorption (continued on next page) J. Catalán et al. Table 1 (continued ) Metabolite Mean (mM) ± SEM (mM) Range (mM) Chemical nature1 KEGG pathways1 Cellular localisation2 PubChem CID2/KEGG entry1 Nucleus Peroxisome Methanol 0.241 ± 0.090 0.043–0.815 Primary alcohol Carbon metabolism Endoplasmic reticulum 887 Degradation of aromatic compounds Extracellular C00132 Metabolic pathways Lysosome Phenylalanine 0.065 ± 0.007 0.036–0.094 Common amino acid Phenylpropanoid biosynthesis – 155,178 Biosynthesis of amino acids C00079 ABC transporters Protein digestion and absorption Proline 0.321 ± 0.036 0.167–0.484 Common amino acid Aminoacyl-tRNA biosynthesis Cytoplasm 145,742 Biosynthesis of amino acids Endoplasmic reticulum C00148 ABC transporters Extracellular Protein digestion and absorption Lysosome Mitochondria Pyruvate 0.198 ± 0.020 0.122–0.299 Carboxylic acids Glycolysis / Gluconeogenesis – 107,735 Citrate cycle (TCA cycle) C00022 Pentose phosphate pathway Biosynthesis of amino acids 131 AMPK signaling pathway Threonine 0.147 ± 0.027 0.033–0.293 Common amino acid Aminoacyl-tRNA biosynthesis Cytoplasm 6288 Biosynthesis of amino acids Extracellular C00188 ABC transporters Protein digestion and absorption Tyrosine 0.076 ± 0.009 0.043–0.120 Common amino acid Aminoacyl-tRNA biosynthesis Cytoplasm 6057 2-Oxocarboxylic acid metabolism Extracellular C00082 Biosynthesis of amino acids Mitochondria Protein digestion and absorption Valine 0.422 ± 0.038 0.258–0.594 Common amino acid Aminoacyl-tRNA biosynthesis Extracellular 6287 2-Oxocarboxylic acid metabolism Mitochondria C00183 Biosynthesis of amino acids ABC transporters Protein digestion and absorption Research in Veterinary Science 153 (2022) 127–136 τ-Methylhistidine 0.059 ± 0.005 0.033–0.082 Modified amino acid β-alanine metabolism Cytoplasm C03298 ABC transporters / GABAergic synapse 64,969 Protein digestion and absorption Histidine Metabolism 1 KEGG Compound Database: https://www.kegg.jp/kegg/compound/. 2 PubChem: https://pubchem.ncbi.nlm.nih.gov/. J. Catalán et al. Research in Veterinary Science 153 (2022) 127–136 Table 2 composition of follicular fluid in donkeys. Yet, several works investi­ Presence and concentration of metabolites identified in jenny and mare pre­ gated the composition of PFF in mares (Fernández-hernández et al., ovulatory follicular fluids (PFF), and in TCM-199 medium. 2020; Gérard et al., 2002; Gérard et al., 2015), the phylogenetically Metabolite Jenny PFF Mare PFF TCM-199 medium closest species, which has been used as a model for applying assisted (mM) (mM)1 (mM)2 reproduction techniques in donkey and the rest of wild equids (Smits 3-Hydroxybutyrate 0.414 – – et al., 2012). A recent report revealed the presence of 22 metabolites in Acetate 1.333 1.500 0.610 mare PFF (Fernández-hernández et al., 2020), in contrast with the 27 Acetlylcarnitine – 0.370 – detected herein in that of jenny (see Table 2). The most interesting Alanine 0.355 1.100 0.280 finding is that only 12 metabolites found in jenny PFF coincided with Aspartate – 2.700 0.220 Betaine 0.106 – – those reported in that of the mare (Fernández-hernández et al., 2020). Carnitine – 0.090 – To note, pyruvate peak was overlapped with that of succinate peak in Choline – 0.030 0.003 the NMR spectrum of mare PFF, these two metabolites being reported Citrate 0.343 0.400 – together in a single concentration (Fernández-hernández et al., 2020). Creatine 0.111 0.360 This did not happen in our study, where the presence of pyruvate in – Creatine phosphate 0,089 0.360 – Creatinine 0.063 – – jenny PFF was confirmed by heteronuclear single quantum correlation. Ethanol 0.794 – – Moreover, concentrations of metabolites found in both mare and jenny Formate 0.815 – – PFF were, except valine, lower in the former than in the latter Fumarate 0.050 – – (Fernández-hernández et al., 2020). These results point to clear differ­ Glucose 4.820 4.300 5.550 Glucose-1- – 6.900 – ences between the metabolic profile of PFF of the jenny compared to the phosphate mare. These variations are attributable to the fact that, despite donkey Glutamine 0.465 – – and horse being phylogenetically close, both species differ for a long Glycerol 0.915 – – time having their own reproductive strategy, which would support that Glycine 0.436 3.200 0.670 the composition of PFF could be species-specific (Canisso et al., 2019). Hippurate 0.053 – – Histamine – 0.250 – Another explanation could be related to the source of PFF samples. In the Histidine – 0.050 0.100 study of mare PFF, the authors mentioned the presence of some intra­ Isoleucine 0.139 0.600 0.300 cellular metabolites, such as glucose 1-phosphate and carnitine Lactate 2.091 27.300 – (Fernández-hernández et al., 2020). The identification of these metab­ Leucine 0.179 0.500 0.460 Lysine 0.131 – – olites in the follicular fluid could be due to release following cell dam­ Methanol 0.241 – – age, as samples were obtained from slaughterhouse ovaries. Cellular Phenylalanine 0.065 – – disruption could also explain the high levels of lactate reported by Proline 0.321 – – Fernández-Hernández et al. (Fernández-hernández et al., 2020), almost Pyruvate 0.198 13 times higher than those observed in our study. The time interval – – Pyruvate + – 0.160 – Succinate between animal death and harvesting of PFF samples could lead to post- Threonine 0.147 0.350 0.250 mortem accumulation of lactate (Tews et al., 1963), as reported by a Tyrosine 0.076 – – previous study carried out with equine oviductal fluid (González- Valine 0.422 0.130 0.210 Fernández et al., 2020). τ-Methylhistidine 0.059 – – TCM-199 is the basic culture medium most used for the formulation 1 Concentration mean of metabolites that were described in mare preovula­ of IVM media in horses and in the attempts carried out so far in donkeys tory follicular fluid (PFF) by Fernández-Hernández et al. (Fernández-hernández (Goudet et al., 2015; Deleuze et al., 2018; Zhao et al., 2011; Abdoon et al., 2020) and concurred with those found in our study in jennies. et al., 2014; Douet et al., 2019). When comparing our results to the 2 Concentration mean of metabolites included in the commercial TCM-199 metabolites described in TCM-199 culture medium (see Table 2), we medium (manufacturer's specifications) that coincided with those found in our detected that only 11 of the 27 metabolites found in jenny PFF were study in jennies. present in TCM-199 medium. In addition, acetate and valine concen­ trations were considerably higher in donkey PFF than in TCM-199 me­ Hinrichs, 2012). For this reason, the very first step for IVP of embryos dium, whereas glycine, isoleucine, leucine, and threonine were less from donors is widely documented in horses: the ultrasound-guided concentrated in the former than in the latter. Although the TCM-199 oocyte retrieval, or Ovum Pick Up (OPU) (Kanitz et al., 1995; Purcell medium is routinely enriched with fetal bovine serum (FBS) to et al., 2007; Jacobson et al., 2010; Galli et al., 2007; Spacek and Car­ perform oocyte IVM (Goudet et al., 2015; Deleuze et al., 2018; Zhao nevale, 2018; Dell'Aquila et al., 1997; Caillaud et al., 2008; Douet et al., et al., 2011; Abdoon et al., 2014; Douet et al., 2019), this supplement 2017). After collection and IVM, oocytes are then fertilized by intra­ only provides eight additional metabolites (Caseiro et al., 2018). Ac­ cytoplasmic sperm injection (ICSI), which in horses is very efficient and cording to the metabolites described in two available commercial FBS the only option, as, to date, conventional IVF does not work in this formulations by Caseiro et al. (Caseiro et al., 2018), supplementation species (Choi and Hinrichs, 2017; Herrera et al., 2015; Jacobson et al., with FBS would provide a series of metabolites. While 18 of these me­ 2010; Galli et al., 2007; Spacek and Carnevale, 2018; Dell'Aquila et al., tabolites are already present in jenny PFF and 7 of them are contained in 1997; Caillaud et al., 2008; Douet et al., 2017; Leemans et al., 2016). TCM-199, 8 identified here in jennies' PFF are missing from TCM-199 + OPU and ICSI are, therefore, established procedures that have been FBS. These metabolites are betaine, citrate, creatine phosphate, creati­ widely implemented to produce horse embryos in vitro, both for scien­ nine, glycerol, hippurate, methanol and pyruvate. Furthermore, three of tific purposes and breeding from infertile and sporting mares (Galli the eight metabolites added through FBS supplementation (isoleucine, et al., 2014). While, as it is one of the key steps for embryo IVP, much leucine, and threonine) are in excess in TCM-199 compared to jenny PFF research has been conducted regarding the IVM of horse oocytes (see Table 2). These differences suggest that the established maturation (Jacobson et al., 2010; Galli et al., 2007; Spacek and Carnevale, 2018; medium used for IVM of horse oocytes (i.e., TCM-199 + FBS) may not be Dell'Aquila et al., 1997; Caillaud et al., 2008; Fernández-hernández suitable for donkeys, which could contribute to explain the poor results et al., 2020; Lewis et al., 2020a; Lewis et al., 2020b; Walter et al., 2019; hitherto obtained (Goudet et al., 2015; Deleuze et al., 2018; Zhao et al., Ascari et al., 2017; Pereira et al., 2019), little has been made in the case 2011; Abdoon et al., 2014; Douet et al., 2019). of donkeys (Zhao et al., 2011). In vitro-matured mare oocytes present significant alterations in the To the best of our knowledge, there are no previous studies about the proteome and metabolome of their cumulus-oophorous complexes 132 J. Catalán et al. Research in Veterinary Science 153 (2022) 127–136 (COCs) in respect to the in vivo-matured (Walter et al., 2019). Among should be taken into account in a future reformulation of IVM media in others, there are differences in the physiological pathways responsible donkeys. for oxygen supply, oxidative phosphorylation and metabolism of Creatine and creatine phosphate constitute the system creatine/ glucose, fatty acids, amino acids, and other substances. This previous phosphocreatine that has the ability to buffer the high-energy phos­ research supports that a physiological approach should be adopted in phate, thus contributing to intracellular energy homeostasis (Jomura the improvement of IVM of oocytes in equids. Formulating IVM media et al., 2021). While both creatine and creatine phosphate are sub­ that brings conditions more similar to the in vivo scenario (Walter et al., products of glycine and arginine metabolism, arginine has not been 2019) could be an option, while another possibility would be adding PFF detected in the PFF of jennies or mares (see Table 2). In human oocytes, to the IVM medium in the case of donkeys. the reduction of arginine in the last six hours of IVM is known to be The metabolites present in the jenny PFF that are missing in routine related to a greater development potential of oocytes (Hemmings et al., IVM media (TCM-199 supplemented with FBS) are pyruvate, citrate, 2013; Houghton et al., 2002). Thus, a possible explanation for the betaine, creatine phosphate, creatinine, glycerol, hippurate, and meth­ absence of arginine in the PFF, but the presence of its products, would be anol. Pyruvate plays a crucial role in oocyte metabolism (Lewis et al., that a high metabolization rate of this amino acid occurs in oocytes that 2020a) and, in bovine, its concentration in the medium is known to are undergoing maturation. In this case, it would be convenient to increase as the oocyte undergoes IVM (Uhde et al., 2018). Follicular maintain the supply of arginine in the culture media, as it does TCM-199 environment in mammalian ovaries relies mainly on anaerobic glycol­ (Fernández-hernández et al., 2020). ysis to satisfy demands of metabolic energy during oocyte maturation, Creatinine levels found in jennies' PFF is considerably lower than consuming glucose and producing lactate as a the end-product (Dumesic those found in mares (see Table 2). While creatinine levels in buffalo FF et al., 2015), which was also found in jenny PFF. In a finely regulated significantly increase at the meta estrous stage compared to the diestrus, cooperation within the COC, glucose is metabolized into pyruvate via these levels do not differ between medium and large sized follicles (Abd glycolysis in the cumulus cells, as the oocyte has low capacity for glucose Ellah et al., 2010). Although creatinine levels in FF have been suggested oxidation because of the lack of phosphofructokinase activity (Cetica to result of an inward movement from blood, the relationship between et al., 2002). Then, this pyruvate is transferred to the oocyte, where it concentration of creatinine in the FF and oocyte viability remains un­ enters mitochondria and is converted into acetyl CoA, which, via the known (Abd Ellah et al., 2010). tricarboxylic acid (TCA) cycle, is used to produce the ATP molecules While this metabolite was not observed in mare's PFF by Fernández- needed for maturation, fertilization and early embryo development Hernández (Fernández-hernández et al., 2020), a considerable concen­ (Dumesic et al., 2015; Cetica et al., 2001; Biggers et al., 1967; Warzych tration of glycerol was detected in jennies' PFF (see Table 2). Glycerol and Lipinska, 2020). Furthermore, pyruvate acts as an antioxidant by was also identified in human PFF, which concentration was correlated neutralizing ROS through its α-keto carboxylate structure (Guarino with the found in blood plasma (Józwik et al., 2007). The biological et al., 2019). In an approach to understand the basic physiology of COC function of glycerol, together with myo-inositol, is to provide the and the effect of in vitro conditions to that physiology, Lewis et al. (Lewis essential alcohol moieties required for the synthesis of cell membrane et al., 2020a) extensively examined in vitro metabolism of equine COC. phospholipids (Fung and Stryer, 1978). Having this key role, it seems They found that adding 0.15 mM pyruvate to the IVM medium led to a preferable that there is bioavailability of glycerol in the IVM media rise in glucose consumption without altering maturation rate, O2 con­ rather than the cell synthesizing glycerol from glucose via glycolysis in sumption or mitochondrial function, suggesting that the current IVM an excess of glucose scenario (Rotondo et al., 2017). systems of equine gametes fail to satisfy the unique metabolic needs of No information on the role of hippurate or methanol in the PFF of the equine oocyte (Lewis et al., 2020a). Thus, while further insights into mares or other species is found in the literature. The concentration of donkey COC metabolic dynamics are needed, the addition of pyruvate at hippurate detected in jenny PFF was the lowest (0.053 mM) from all the an adequate concentration for donkey IVM media should be envisaged. metabolites. Although this low value points to a residual importance of On the other hand, citrate is an intermediate metabolite produced hippurate, it could have some specific role in jenny PFF. Methanol, as during glucose/pyruvate metabolism in the TCA cycle (Fernández- indicated in Table 1, is a subproduct of either carbon metabolism or the hernández et al., 2020). Citrate acts as a linkage between CTA cycle, degradation of aromatic compounds. As methanol was not found in mare β-hidroxibutirate and lipid metabolism in FF (Piñero-Sagredo et al., PFF, one could suggest that this metabolite has a particular significance 2010; Guo et al., 2018). Furthermore, citrate has been found to act as a in jennies. key substrate epigenetic modifications in the oocyte (Gu et al., 2015; It is worth mentioning that, among the 27 metabolites identified in Harvey, 2019), while it seems to be play an important role during early jennies PFF, almost 41% were common amino acids (alanine, phenyl­ embryogenesis, as a positive correlation between citrate concentration alanine, glutamine, glycine, isoleucine, leucine, lysine, proline, threo­ in PFF and the number of embryos produced per matured oocyte in nine, tyrosine, and valine), and around 15% were modified amino acids humans (Castiglione Morelli et al., 2019). He et al. (He et al., 2022) or amino acid derivatives (creatine, creatine phosphate, τ-methyl­ recently revealed that the adverse impact of autophagy impairment on histidine and betaine) (See Fig. 1). The biological functions of these oocyte quality is mediated by downregulated citrate levels, which once biomolecules in the mammalian oocyte are diverse. In addition to being enhanced by exogenous supplementation could significantly restore basic substrates for protein synthesis (Collado-Fernandez et al., 2013), oocyte maturation. They also observed that the decreased autophagy which is very active during oocyte maturation (Tomek et al., 2002), levels in cumulus cells, typical of maternal aging, are related to lower amino acids play important roles in cell function (Sturmey et al., 2008), levels of citrate in human PFF, and that elevated citrate levels in porcine such as energy substrates and regulators of osmolarity and pH (Collado- PFF can promote oocyte maturation (He et al., 2022). Therefore, the lack Fernandez et al., 2013). In this sense, it has been reported that the amino of citrate in the IVM conditions currently used in donkeys should be acid profile is a good predictor of oocyte development competence and addressed by incorporating it in the media. embryo quality (Collado-Fernandez et al., 2013). In this study, we could Betaine (N,N,N-trimethylglycine) is an amino acid derivative that observe glutamine, glycine and alanine as the most concentrated amino has two known important roles in mammals: cell volume homeostasis, acids present in donkey PFP. Glutamine and glycine have been reported which has been proven in mouse embryos, and to be a source of methyl to be required for de novo synthesis of purine and pyrimidine nucleotides groups, becoming a key methyl donor in blastocysts (Corbett et al., for mRNA synthesis (Collado-Fernandez et al., 2013). In bovine FF, 2014). Supplementation of IVM media with 16 mM betaine significantly contrary to the fatty acids profile that did not have predictive value, boosts the levels of nucleic acid N6-methyladenosine epigenetic modi­ some amino acids have been identified as useful markers of the oocyte in fication, which is important for meiotic maturation (Wang et al., 2018), vitro competence, as their presence in the PFF was associated with good in porcine COCs. Based on the physiological importance of betaine, it quality morphological aspect and subsequent development until the 133 J. Catalán et al. Research in Veterinary Science 153 (2022) 127–136 3.70% Investigation: J.C., I.M.-R.; I.Y.-O.; Y.M.-O.; A.B.-F.; P.N. and J.M.; Methodology, J.C., I.M.-R.; I.Y.-O.; Y.M.-O.; A.B.-F.; M.Y. and J.M.; 11.11% Project administration, M.Y. and J.M.; Resources, P.N.; M.Y. and J.M.; 14.81% Supervision, M.Y. and J.M.; Writing - original draft: J.C. and I.M.; Fatty acids Writing - review & editing: M.Y. and J.M. Amino acid derivatives P.N. NMR data acquisition and analysis. 3.70% Saccharides Carboxylic acids Declaration of Competing Interest Lactams 40.74% 22.22% Common amino acids The authors declare that there is no conflict of interest that could be Alcohols perceived as prejudicing the impartiality of the research reported herein. Acknowledgments 3.70% J.C. was funded by the project “Demetra” (Dipartamente di Eccel­ Fig. 1. Chemical nature of the 27 metabolites found in jennies preovulatory lenza 2018–2022, CUP_C46C18000530001), Italian Ministry for Edu­ follicular fluid (PFF), and proportion of metabolites found in jennies PFF ac­ cation, University and Research. I.M.-R. was awarded a scholarship from cording to their chemical nature. the Spanish Ministry of Science, Innovation and Universities (BES-2017- 081962). I⋅Y.-O. was funded by the Secretary of Higher Education, blastocysts stage (Sinclair et al., 2008; Matoba et al., 2014). Likewise, in Science, Technology and Innovation (SENESCYT), Ecuador (Scheme: cattle, the concentrations of leucine and cysteine in the follicular fluid Programa de Becas Internacionales de Posgrado 2019; Grant: CZ02- have been related to in vivo fertility (Moore et al., 2017). 000507-2019). The authors also acknowledge the support from the The metabolic profile of donkey PFF reported in this study is the first Ministry of Science and Innovation, Spain (Grant: AGL2017-88329-R), attempt to address that composition in this species, while also trying to the Catalan Agency for Management of University and Research Grants, optimize the media for IVM of oocyte. Our results showed clear differ­ Regional Government of Catalonia, Spain (Grant: 2017-SGR-1229); and ences in the metabolites found in jenny PFF compared to those found in the Catalan Institution for Research and Advanced Studies, Spain that of the mare, and to those of the maturation medium composed by (ICREA). TCM-199 supplemented with FBS. These divergences indicate that both equine species differ in the composition of their PFF and point to the Appendix A. Supplementary data scope for improvement in the IVM media used up to now in the jenny, towards the optimization of IVM rates and obtaining IVP embryos, Supplementary data to this article can be found online at https://doi. which has not yet been achieved in donkeys. Further research should be org/10.1016/j.rvsc.2022.10.026. focused on elucidating the PFF metabolome across the different follic­ ular stages, to learn more about the dynamics during the maturation process in vivo. It is also important to consider that the metabolites are References only part of the different components of PFF, such as hormones, pro­ Abd Ellah, M.R., Hussein, H.A., Derar, D.R., 2010. 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