The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.

With the rising global demand for seafood and the challenges posed by overfishing and climate change, the aquaculture sector has become increasingly important in providing high‐quality protein for human consumption. Although traditional selection breeding programs have made great strides in genetic improvement of aquaculture species over the past decades, faster and more precise breeding tools, such as genome manipulation, are needed for performance enhancement of aquaculture stock. This review presents a comprehensive overview of the current status of three major genome manipulation tools, including RNA interference (RNAi), gene transfer, and genome editing in aquaculture species, and discusses the advances made, challenges faced, and potential future directions of this fast‐developing field. Taking catfish as an example, this paper reviews the specific applications of these techniques to improve traits such as growth, disease resistance, reproduction, and nutritional profiles in various commercially important fishes and crustaceans, highlighting successful applications and ongoing research efforts. We also propose CRISPR/Cas9‐mediated multiplex genome editing for the knockout or replacement of multiple genes in parallel to improve multiple traits in fish. Collectively, this review provides insights into the evolving landscape of genome manipulation in aquaculture and sheds light on its implications for sustainable practices and responsible innovation.

The aim of the present study was to investigate the spontaneous motility of spermatozoa and to optimize sperm collection, short-term sperm storage, and fertilization in zebrafish Danio rerio. The movement of spermatozoon in water was propagated along the flagellum at 16 s after sperm activation then damped from the end of the flagellum for 35 s and fully disappeared at 61 s after activation. For artificial fertilization, milt must be added to an immobilizing solution, which stops the movement of sperm and keeps the sperm motionless until fertilization. E400 and Kurokura as isotonic solutions were shown to be suitable extenders to store sperm for fertilization for 6 h. E400 stored sperm for 12 h at 0–2 °C. Sperm motility decreased only to 36% at 12 h post stripping for the E400 extender and to 19% for the Kurokura extender. To achieve an optimal level of fertilization and swim-up larvae rates, a test tube with a well-defined amount of 6,000,000 spermatozoa in E400 extender per 100 eggs and 100 µL of activation solution has proven to be more successful than using a Petri dish. The highest fertilization and swim-up larvae rates reached 80% and 40–60%, respectively, with milt stored for 1.5 h in the E400 extender at 0–2 °C.