Besides allowing an improvement of animal reproductive efficiency, assisted reproductive technologies (ARTs) have become an important tool for biodiversity preservation [1,2]. More and more species are threatened with extinction or experiencing a decrease in population size. Germplasm cryopreservation, and especially gamete cryopreservation, has become a crucial part of biodiversity preservation programs, since it allows to store genetic material for future use and to plan breeding with animals that are distant both in time and space. Among the most endangered mammalian taxa are the felids, and for some of them, such as the Iberian lynx, some conservation programs have already been implemented successfully [3,4]. However, assisted reproduction protocols are not efficient for most endangered species [5] and the use of closely related domestic species is useful to design specific procedures to be transferred to the threatened ones. For felids, the domestic cat is an excellent model [6].

Among germplasm cryopreservation options, gamete banking is probably the one that offers the best balance between efficiency and flexibility. Gonadal tissue preservation could offer the chance to store abundant germ cells, but it is still technically challenging, especially with regards to development of such gamete precursors into mature gametes after thawing, either in vivo post grafting or in vitro after culture [7]. Instead, gamete banking already proved to be successful, to some extent. In addition, storing male and female gamete separately offers the chance to better exploit genetic diversity and plan appropriately for population management in the future [8], while, for instance, embryo cryopreservation does not allow the same flexibility in the generation of offspring, since the combination of the male and female gametes has already been decided. Male gametes can be retrieved from ejaculates from living subjects, or from isolated gonads, and in particular from the cauda epididymis in case of castration or death, but this topic lies outside the scope of the present work. Female gametes, instead, can be surgically retrieved from living animals (also after hormonal stimulation) or from isolated ovaries after spaying or death. Most of the times, the animals are not hormonally stimulated, especially if the gamete collection takes place from isolated ovaries. In this case, it is unlikely to obtain mature (metaphase II, MII) oocytes, which would be ready to be fertilized, therefore, immature (germinal vesicle, GV) oocytes are usually retrieved. The GV oocytes can be either matured in vitro and then cryopreserved or they can be cryopreserved as GV right after collection. Efficiency of cryopreservation at different stages of oocyte maturation can vary, as described below.

The most used cryopreservation techniques, for oocytes, are slow (or controlled rate) freezing and vitrification. Although vitrification was developed later, it gained popularity for cat oocytes during the years (Fig. 1) thanks to its speed, ease of application and field-feasibility. Both techniques, however, currently do not allow to achieve satisfactory results. After freezing-thawing or vitrification-warming, oocyte struggle to mature and/or develop into embryos. In vitro maturation (IVM) after warming is a challenge for the GV oocytes, and cleavage after in vitro fertilization (IVF) usually reaches 15–30% [[9], [10], [11], [12], [13], [14], [15]] (Table 1), while for mature cryopreserved gametes it can reach 30–50% [[16], [17], [18]] (Table 2). However, progression of embryos to late in vitro stages is often impaired [19] (Table 1, Table 2), and improvements to cryopreservation protocols are needed. Even if the viability after warming is usually as high as 90% [19,20], many oocytes degenerate during the following in vitro culture, but the mechanisms causing injuries are still largely unclear [21]. Different approaches were experimented, such as acting on the oocytes prior to cryopreservation or using chemical or physical enrichment during the cryopreservation procedure itself or after thawing or warming, nonetheless maturation and embryonic developmental rates remained poor compared to fresh oocytes.

The purpose of this paper is to review cat oocyte vitrification, the strategies attempted so far to improve the outcomes and the time for their application (i.e., before, during, or after vitrification-warming), as well as analyzing other possible approaches for future trials.