Microenvironment factors promoting the quality of vitrified cat oocytes

In oocyte cryopreservation programs, vitrification has overthrown conventional slow freezing both in veterinary and human medicine. In animals, its feasibility in field conditions makes it the preferred technique for the safeguard of genetic resources from zoo or wild animals, including threatened felids, for which the domestic cat is an outstanding model. The avoidance of ice formation saves oocytes from a certain extent of damage, but other cellular injuries, such as cytoskeleton, mitochondria and meiotic spindle alterations, DNA damage, zona pellucida hardening and cumulus cell loss, might occur as a consequence of vitrification. After warming, although the exact mechanisms are still unclear, degeneration is a frequent outcome for cat vitrified oocytes. For immature (germinal vesicle) gametes, in vitro maturation after warming is a challenge, and cleavage after fertilization barely reaches 15-30%, while for mature (metaphase II) cryopreserved gametes it can get to 30-50%. Anyway, the progression to late embryos stages is often impaired, and improvements are needed. Despite being necessary to achieve the vitrification state, the exposure to high concentrations of cryoprotectants and the sharp decrease of temperature are far from physiological conditions, and after warming the conventional in vitro culture may not be enough for vitrified oocytes to recover and demonstrate their full developmental potential. Physical or chemical enrichments to the culture microenvironment could create more favorable conditions and promote vitrified oocyte survival and embryo development. To better preserve intracellular architecture, our recent studies employed three-dimensional (3D) culture systems for the in vitro maturation of vitrified oocytes. The 3D system, together with companion cells, was able to support meiotic progression, but the physicochemical-enriched environment was not enough to obtain a significant change in vitrified oocyte developmental abilities [1]. The use of a different physical modification of the culture environment (i.e., 3D liquid marble microbioreactor), also led to similar results. Following a different approach, some studies focused their attention on specific intracellular pathways. For instance, the chemical inhibition of apoptosissignaling molecules to prevent cell death [2,3] gave encouraging results for what concerns in vitro maturation and embryo development. Although continuous progresses are being made, there is still a strong need to enhance feline vitrified oocytes outcomes. When specific genetic pools have to be preserved or when there is no male counterpart for fertilization, oocyte cryopreservation is the only possibility, and the development of new vitrification and culture strategies will be crucial. Supported by Polish National Agency for Academic Exchange, Grant PPI/APM/2019/1/00044/U/00001 and by Università degli Studi di Milano "Piano di Sostegno alla Ricerca 2020 (Linea 2 Azione A). [1] Colombo M, Morselli MG, Tavares MR, et al. Developmental competence of domestic cat vitrified oocytes in 3D enriched culture conditions. Animals 2019;9:329. [2] Arayatham S, Tiptanavattana N, Tharasanit T. Effects of vitrification and a Rho-associated coiledcoil containing protein kinase 1 inhibitor on the meiotic and developmental competence of feline oocytes. J Reprod Dev 2017;63:511-7. [3] Colombo M, Zahmel J, Jänsch S, et al. Inhibition of apoptotic pathways improves DNA integrity but not developmental competence of domestic cat immature vitrified oocytes. Frontiers in Veterinary Science 2020;7:766.