Cryobiology xxx (xxxx) xxx Please cite this article as: Irene Menéndez-Blanco, Cryobiology, https://doi.org/10.1016/j.cryobiol.2020.02.011 Available online 25 February 2020 0011-2240/© 2020 Published by Elsevier Inc. Effect of vitrifcation of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection Irene Men� endez-Blanco a , Sandra Soto-Heras a , María G. Catal� a a , Anna-Rita Piras b , Dolors Izquierdo a , María-Teresa Paramio a, * a Universitat Aut� onoma de Barcelona, Departament de Ci� encia Animal i Dels Aliments, 08193, Bellaterra, Barcelona, Spain b Universit� a Degli Studi di Sassari, Dipartimento di Medicina Veterinaria. V.Vienna 2, 07100, Sassari, Italy A R T I C L E INFO Keywords: Goat IVM-Oocytes Prepubertal Vitrifcation ABSTRACT This work studies the effect of vitrifcation of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22–24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrifed/warmed IVM-oocytes were vitrifed after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (Me 2 SO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 μM 2 0 7 0 dichlorodihydrofuorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classifed as: Live (AV  PI  ), early apoptotic (AV þ PI  ), dead non-apoptotic (AV  PI þ ) and necrotic (AV þ PI þ ). Developmental competence of vitrifed/warmed oocytes was assessed by PA (5 min in 5 μM Ionomycin plus 4 h in 2 mM 6-Dime- thylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrifed/warmed oocytes showed higher ROS levels (P < 0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differ- ences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, signifcant differences were found in cleavage rate (59 vs.78%; P < 0.0343) and blastocyst formation (1 vs. 25%; P < 0.0001). In conclusion, vitrifcation reduced oocyte competence by increasing dead oocytes and ROS levels. 1. Introduction Nowadays vitrifcation is the most useful technique for the cryo- preservation of oocytes and embryos. Cryopreservation of oocytes has been very troublesome comparing with sperm or embryo cryopreser- vation. The widely use of vitrifcation for cryopreservation of oocytes and embryos could be explained by the decreased risk of damage caused by the lack of ice crystal formation associated to this technique [20]. Vitrifcation techniques are mainly based on very higher concentrations of cryprotectants that could reduce the formation of ice inside the cell. Vitrifcation leads the water to a solidifed but not crystallized state creating a glasslike system. This generates such high level of toxicity that the cell can be only exposed for very short period of time in a minimum volume of solutions [4]. In the last years several vitrifcation protocols have been described, which differ from one another in the type of cryoprotectants (ethylene glycol [EG] and/or dimethyl sulfoxide [Me 2 SO] and/or propylene glycol [PROH] and sucrose and/or Ficoll and/or trehalose), equilibration and dilution parameters, the carrier tools and the cooling, storage and warming methods (reviewed by Ref. [33]). To date the most commonly used protocol for oocyte vitrifcation involves the combination of 15% Me 2 SO, 15% EG and 0.5 M sucrose in a minimum volume (�1 μl) [17, 18]. The oocyte is a large and susceptible cell for cryopreservation * Corresponding author. Departament de Ci� encia Animal i dels Aliments, Facultat de Veterinaria. Universitat Aut� onoma de Barcelona, 08193, Bellaterra, Barcelona, Spain. E-mail address: teresa.paramio@uab.es (M.-T. Paramio). Contents lists available at ScienceDirect Cryobiology journal homepage: http://www.elsevier.com/locate/cryo https://doi.org/10.1016/j.cryobiol.2020.02.011 Received 5 December 2019; Received in revised form 21 February 2020; Accepted 24 February 2020