Sturgeons (Acipenseriformes) are one of the most critically endangered groups of species, necessitating urgent and effective measures to conserve their remaining diversity. However, the development of efficient techniques for the cryopreservation of fish eggs and embryos has been particularly challenging due to the large size of the oocyte, high yolk content, and low permeability to cryoprotectants [1,2]. As a result, maternal genetics, represented primarily by mitochondria, cannot currently be successfully preserved. This limitation has driven the exploration of alternative methods to secure matrilineal genetic information.

Sturgeons exhibit a unique mode of germline development that makes them an exceptional model for studying germ plasm and its components independently of other embryonic lineages. In sturgeon embryos, germ plasm is localized exclusively at the vegetal pole, where PGCs are formed, while the remaining vegetal hemisphere is utilized as endogenous nutrition in the form of yolky cells [[3], [4], [5]]. This spatial separation allows for targeted experimental manipulation of germ plasm without affecting other somatic lineages. Building on this unique developmental architecture, we developed a novel method for the elimination of germ plasm including mitochondria using UV irradiation [6]. In turn we transplanted exogenous mitochondria specifically into the germplasm and they rescued PGCs formation. Transplantation of egg-derived mitochondria fully restored the function of the germ plasm and enabled the development of primordial germ cells (PGCs). These mitochondria were subsequently incorporated into the germline [7]. These findings highlight the necessity of optimizing cryopreservation methods to ensure the availability of healthy, functional mitochondria for such conservation techniques.

While mitochondria have been isolated and cryopreserved since the 1970s, using cryoprotectants such as glycerol and dimethyl sulfoxide (DMSO) [8], this technology remains underdeveloped, with varying degrees of success depending on the species and protocol used [9]. Cryopreservation, freezing, and thawing processes often disrupt mitochondrial structural and functional integrity, limiting their usability in research and applications [[10], [11], [12], [13]].

In parallel, alternative approaches have sought to address the challenges of cryopreserving fish eggs and embryos by focusing on germline stem cells, such as embryonic PGCs or adult gonial stem cells [14,15]. These cells can be isolated from a target species and stored in liquid nitrogen, preserving their functionality for future use. Upon thawing, they can be transplanted into surrogate parents with depleted germ cells to produce donor-derived progeny [[16], [17], [18]]. This biotechnology has shown success in several fish species, including advancements in optimizing gonial stem cell transplantation in interspecies recipients for sturgeon [19]. Methods to eliminate PGCs, such as antisense morpholino oligonucleotides [20], CRISPR/Cas9 gene editing targeting the dnd1 gene [21], and UV irradiation targeting the germ plasm [6], have further refined these techniques. However, despite significant progress, production of donor-derived progeny in sturgeons has yet to be achieved [[22], [23], [24]].

Only a few studies have focused on the cryopreservation of isolated mitochondria, and the strategies employed vary widely across different studies [8,9,[25], [26], [27]]. To date, no studies on the cryopreservation of isolated sturgeon egg mitochondria have been identified and no protocols are available, underscoring the necessity for optimized methodologies in this context. Therefore, the present study aimed to preserve isolated sturgeon egg mitochondria for extended periods without compromising their structural and functional integrity. Furthermore, we sought to evaluate their capacity for transplantation and their ability to rescue PGCs.