animals Article New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification Daniel Angel-Velez 1,2, * , Tine De Coster 1,† , Nima Azari-Dolatabad 1 , Andrea Fernandez-Montoro 1 , Camilla Benedetti 1 , Osvaldo Bogado Pascottini 1,3 , Henri Woelders 4 , Ann Van Soom 1 and Katrien Smits 1   Citation: Angel-Velez, D.; De Coster, T.; Azari-Dolatabad, N.; Fernandez-Montoro, A.; Benedetti, C.; Bogado Pascottini, O.; Woelders, H.; Van Soom, A.; Smits, K. New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification. Animals 2021, 11, 3077. https://doi.org/10.3390/ ani11113077 Academic Editor: Olga García-Álvarez Received: 30 September 2021 Accepted: 26 October 2021 Published: 28 October 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; tine.decoster@ugent.be (T.D.C.); nima.azaridolatabad@ugent.be (N.A.-D.); andrea.fernandezmontoro@ugent.be (A.F.-M.); camilla.benedetti@ugent.be (C.B.); osvaldo.bogado@ugent.be (O.B.P.); ann.vansoom@ugent.be (A.V.S.); katrien.smits@ugent.be (K.S.) 2 Research Group in Animal Sciences—INCA-CES, Universidad CES, Medellin 050021, Colombia 3 Veterinary Physiology and Biochemistry, Departmentof Veterinary Sciences, University of Antwerp, 2610 Wilrijk, Belgium 4 Wageningen Livestock Research, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands; Henri.Woelders@wur.nl * Correspondence: daniel.angelvelez@ugent.be or danielangel30@gmail.com; Tel.: +32-4-5614-4543 † Daniel Angel-Velez and Tine De Coster should be considered joint first authors. Simple Summary: Oocyte cryopreservation allows female gametes to be conserved for long periods, which would be of benefit for mares of high genetic merit, but its efficiency is not satisfactory yet. Therefore, the aim of this study was to optimize a vitrification protocol for equine oocytes using a systematic approach. We performed a side-by-side comparison of different cryoprotective agents (CPAs) during the vitrification and warming of equine oocytes. In the first experiment, a fixed mixture of CPAs that enter the oocyte was used, and three sugars were compared, which cannot penetrate the oocyte but provide protection through an osmotic effect. In the second experiment, one sugar from the first experiment was selected to compare three mixtures of CPAs that enter the oocyte. Overall, the embryo development was reduced after oocyte cryopreservation when compared to fresh oocytes. Yet, we were able to produce embryos with all six cryoprotective agent mixtures, and we identified one promising combination of cryoprotectants, consisting of propylene glycol, ethylene glycol, and galactose, that resulted in blastocyst rates in the same range as the fresh control group. Abstract: Equine oocyte vitrification would benefit the growing in vitro embryo production programs, but further optimization of the protocol is necessary to reach clinical efficiency. Therefore, we aimed to perform a direct comparison of non-permeating and permeating cryoprotective agents (CPAs) during the vitrification and warming of equine immature oocytes. In the first experiment, cumulus oocytes complexes (COCs) were vitrified comparing sucrose, trehalose, and galactose in combination with ethylene glycol (EG) and dimethyl sulfoxide (DMSO). In the second experiment, the COCs were vitrified using three mixtures of permeating CPAs in a 50:50 volume ratio (ethylene glycol-dimethyl sulfoxide (ED), propylene glycol-ethylene glycol (PE), and propylene glycol-dimethyl sulfoxide (PD)) with galactose and warmed in different galactose concentrations (0.3 or 0.5 mol/L). Overall, all the treatments supported blastocyst formation, but the developmental rates were lower for all the vitrified groups in the first (4.3 to 7.6%) and the second (3.5 to 9.4%) experiment compared to the control (26.5 and 34.2%, respectively; p < 0.01). In the first experiment, the maturation was not affected by vitrification. The sucrose exhibited lower cleavage than the control (p = 0.02). Although the galactose tended to have lower maturation than trehalose (p = 0.060) and control (p = 0.069), the highest numerical cleavage and blastocyst rates were obtained with this CPA. In the second experiment, the maturation, cleavage, and blastocyst rates were similar between the treatments. Compared to the control, only the ED reached similar maturation (p = 0.02) and PE similar cleavage (p = 0.1). The galactose concentration during warming did not affect the maturation, cleavage, or blastocyst rates (p > 0.1), but the PE-0.3 exhibited the highest blastocyst rate (15.1%) among the Animals 2021, 11, 3077. https://doi.org/10.3390/ani11113077 https://www.mdpi.com/journal/animals