Aquaculture is the fastest‐growing food‐production sector and is striving to become a long‐term sustainable approach to meet the rising global demand for seafood. During the expansion and advancement of aquaculture, minimizing ecological impacts should occur concomitantly with maximizing production. Farmed fish, often genetically distinct from their natural conspecifics, may pose significant risks of genetic contamination and ecological imbalance to wild populations if they escape from aquaculture confinement. Growing reproductively sterile fish is the most effective way to genetically contain farmed fish. Atlantic salmon (Salmo salar) escape events in the ‘Pacific Northwest’ region of the United States and Canada have raised alarms over potential ecological impacts and led to legislation in Washington State phasing out the culture of non‐native finfish species. Farming sterile native species such as coho salmon (Oncorhynchus kisutch) and sablefish (Anoplopoma fimbria) in the Pacific Northwest would ease public concerns and promote environmentally and economically sustainable aquaculture. Sterile fish also can mitigate the challenge of precocious maturation, a prominent issue associated with culture of salmonids and many other species, to improve somatic growth, flesh quality and fish health and welfare. Here, we review methods having potential applications for producing sterile fish and introduce our novel immersion‐based technology that temporarily silences the dead end (dnd) gene using Morpholino oligonucleotides to produce sterile coho salmon and sablefish for the first time. The successful induction of sterility in these two iconic Pacific Northwest species without introducing genetic modifications would promote the use of this immersion‐based sterilization technology for more aquaculture finfish worldwide.

Germ cell transplantation technology enables surrogate offspring production in fish. This technology has been expected to mitigate reproductive barriers, such as long generation time, limited fecundity, and complex broodstock management, enhancing seed production and productivity in aquaculture. Many studies of germ cell transplantation in various fish species have been reported over a few decades. So far, surrogate offspring production has been achieved in many commercial species. In addition, the knowledge of fish germ cell biology and the related technologies that can enhance transplantation efficiency and productivity has been developed. Nevertheless, the commercial application of this technology still seems to lag behind, indicating that the established models are neither beneficial nor cost-effective enough to attract potential commercial users of this technology. Furthermore, there are existing bottlenecks in practical aspects such as impractical shortening of generation time, shortage of donor cells with limited resources, low efficiency, and unsuccessful surrogate offspring production in some fish species. These obstacles need to be overcome through further technology developments. Thus, we thoroughly reviewed the studies on fish germ cell transplantation reported to date, focusing on the practicality, and proposed potential solutions and future perspectives.

Fish ovarian germline stem cells (OGSCs) have great potential in various biological fields due to their ability to generate large numbers of mature eggs. Therefore, selective enrichment of OGSCs is a prerequisite for successful applications. To determine the optimal conditions for the enrichment of OGSCs from Japanese medaka (Oryzias latipes), we evaluated the effects of Percoll density gradient centrifugation (PDGC), differential plating (DP), and a combination of both methods. Based on cell morphology and gene expression of germ cell-specific Vasa and OGSC-specific Nanos2, we demonstrated that of seven density fractions obtained following PDGC, the 30–35% density fraction contained the highest proportion of OGSCs, and that Matrigel was the most effective biomolecule for the enrichment of Oryzias latipes OGSCs by DP in comparison to laminin, fibronectin, gelatin, and poly-l-lysine. Furthermore, we confirmed that PDGC and DP in combination significantly enhanced the efficiency of OGSC enrichment. The enriched cells were able to localize in the gonadal region at a higher efficiency compared to non-enriched ovarian cells when transplanted into the developing larvae. Our approach provides an efficient way to enrich OGSCs without using OGSC-specific surface markers or transgenic strains expressing OGSC-specific reporter proteins.

Although testicular organoids have remarkable potential as testicular models in vitro, there have been few studies about testicular organoids in teleost fish. As a first step to establish a stable culture system for fish testicular organoids, we investigated the efficient conditions for an aggregate culture of dispersed testicular cells from adult marine medaka (Oryzias dancena) by evaluating the effects of culture methods and media composition on an aggregate culture. As the results, we found that culturing dispersed testicular cells in an ultra-low attachment 96 well without Matrigel was most effectively able to induce the formation of testicular cell aggregates among the five different methods tested. Subsequently, through media testing, we confirmed that the modified ESM2 was more optimal for this aggregate culture than the media conventionally used in porcine, human, and rat testicular aggregate cultures. Furthermore, we demonstrated that three supplements in the modified ESM2 including fish serum (FS), basic fibroblast growth factor (bFGF), and embryo extracts (EE) did not influence the number and size of the testicular aggregates formed, but fetal bovine serum and other supplements including β-mercaptoethanol, non-essential amino acids, sodium pyruvate, and sodium selenite were affected significantly. Nevertheless, the removal of three supplements (FS, bFGF, and EE) during culture negatively affected scp3 and sox9a expression levels, indicating their necessity. Finally, we identified that the sperms derived from in vitro cultured testicular aggregates were able to produce offspring after fertilization with naturally matured oocytes. The results from this study will provide fundamental information to develop the techniques for fish testicular organoid culture, which will eventually contribute to the development of reproductive biotechnology for aquaculture and the conservation of endangered fish species.

Development of advanced cell culture methods has gained increasing attention because it allows for efficient genetic engineering and precise regulation of animal reproduction on a cellular basis. Numerous studies have attempted to develop an advanced cell culture method. Previous studies have altered cell culture media and pretreated culture plates with functional molecules. Among them, a mussel-inspired polymer coating has been extensively utilized owing to its wide applicability. For instance, adhesion of human embryonic stem cells and neuronal cells on solid surfaces has been improved. Despite the excellent capability of the mussel-inspired polymer coating, most studies have primarily focused on mammalian cells. However, the efficacy of these coatings on the adhesion of other cell lines is yet unclear. This study aimed to assess the potential of the mussel-inspired polymer coating in the regulation of the adhesion of fish ovarian germline stem cells on solid surfaces. Solid surfaces were coated by polydopamine and poly-L-lysine, and the effect of the coatings on cellular behaviors was investigated.