Various antioxidants are tested to improve the viability and development of cryopreserved oocytes, due to their known positive health effects. The aim of this study was to find whether astaxanthin (AX), a xanthophyll carotenoid, could mitigate deteriorations that occurred during the vitrification/warming process in bovine oocytes. Astaxanthin (2.5 µM) was added to the maturation medium during the post-warm recovery period of vitrified oocytes for 3 h. Afterward, the oocytes were fertilized in vitro using frozen bull semen and presumptive zygotes were cultured in the B2 Menezo medium in a co-culture with BRL-1 cells at 38.5 °C and 5% CO2 until the blastocyst stage. AX addition significantly reduced ROS formation, lipid peroxidation, and lysosomal activity, while increasing mitochondrial activity in vitrified oocytes. Although the effect of AX on embryo development was not observed, it stimulated cell proliferation in the blastocysts derived from vitrified oocytes and improved their quality by upregulation or downregulation of some genes related to apoptosis (BCL2, CAS9), oxidative stress (GPX4, CDX2), and development (GJB5) compared to the vitrified group without AX. Therefore, the antioxidant properties of astaxanthin even during short exposure to bovine vitrified/warmed oocytes resulted in improved blastocyst quality comparable to those from fresh oocytes.

Department of Botany and Genetics Faculty of Natural Sciences and Informatics Constantine the Philosopher University in Nitra Tr A Hlinku 1 949 01 Nitra Slovakia

Institute of Biotechnology Faculty of Biotechnology and Food Science Slovak Agricultural University in Nitra Tr A Hlinku 2 949 76 Nitra Slovakia

Laboratory of Developmental Biology Institute for Animal Physiology Genetics of the Czech Academy of Sciences Rumburská 89 277 21 Liběchov Czech Republic

Research Institute for Animal Production Nitra National Agricultural and Food Centre Hlohovecká 2 951 41 Lužianky Slovakia

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Reactive oxygen and nitrogen species: multifaceted regulators of ovarian activity†