Conservation programmes for wild and cultured fish have been established worldwide in order to protect them from becoming extinct [9]. Cryopreservation of aquatic germplasm brings the possibility of preserving the genome of endangered species, increasing the representation of genetically valuable animals for farming purposes and avoiding genetic losses through diseases and catastrophes [3] and [9]. For majority of animal species, cryopreservation of embryos at any developmental stages still represents

major challenges. Whereas, according to Saragusty and Arav [29], thousands of offspring were born following the transfer of frozen-thawed embryos in humans, cattle and mice, success is very limited in many others, even closely related species. In fish, successful cryopreservation of semen from many species including salmonids, cyprinids, cichlids, silurids, acipenserids, anastomids and E7080 supplier characids has been well documented [4], [6], [7], [37] and [38] and cryopreserved semen has been used for reproduction of many wild and farmed species [11]. Attempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation

protocol for long-term storage still remains elusive [5], [8], [14], [15], [47] and [48]. Fish embryos are multi-compartmentalized, and there are several barriers that have been identified as obstacles for successful cryopreservation: their high yolk content, large size, low permeability of the membranes and their high sensitivity to chilling [26]. It has recently been reported [39] that the use AZD2281 mouse of oocytes may offer some advantages when compared to fish embryos, mainly due to the absence of a Unoprostone fully formed chorion, their smaller size resulting

in higher surface-to-volume ratio and higher membrane permeability, therefore improving the chances of successful cryopreservation. Although several studies have been carried out on fish oocytes cryopreservation [16], [21], [23], [24] and [39], all of them used controlled slow cooling protocol and success remains elusive as for embryos. Only one study, carried out by Guan et al. [12] reported the use of vitrification for isolated stage III zebrafish ovarian follicles, however the ovarian follicles were severely damaged during the process. Despite the successful use of vitrification technique for oocytes cryopreservation in humans [19] and some domestic mammals [41], very limited studies on vitrification of fish oocytes has been carried out to date. Vitrification is an ice-free cryopreservation method using high concentrations of cryoprotectants (CPAs) and ultra-rapid cooling rates [25] which offers advantages that may contribute to overcome some of the difficulties associated with the slow cooling protocols. The present study aimed to develop a cryopreservation protocol for stage III zebrafish ovarian follicles in tissue fragments using vitrification.