Sturgeons also known as living fossils are facing threats to their survival due to overfishing and interference in natural habitats. Sterlet (Acipenser ruthenus) due to its rapid reproductive cycle and small body size can be used as a sterile host for surrogate production for late maturing and large sturgeon species. Dead end protein (dnd1) is essential for migration of Primordial Germ Cells (PGCs), the origin of all germ cells in developing embryos. Knockout or knockdown of dnd1 can be done in order to mismigrate PGCs. Previously we have used MO and UV for the aforementioned purpose, and in our present study we have used CRISPR/Cas9 technology to knockout dnd1. No or a smaller number of PGCs were detected in crispants, and we also observed malformations in some CRISPR/Cas9 injected embryos. Furthermore, we compared three established methods to achieve sterility in sterlet, and we found higher embryo survival and hatching rates in CRISPR/Cas9, UV and MO, respectively.

We report for first time comparison of two approaches for zebrafish triploid production using cold shock and heat shock treatment. Subsequently, produced triploid zebrafish were used as a recipients for intraperitoneal transplantation of ovarian and testicular cells originating from vas:EGFP strain in order to verify their suitability for surrogate reproduction. Heat shock treatment was far more effective evaluated as success rate of triploid production and viability in comparison to cold shock.Triploids were produced with up to 100% efficiency in particular females. As expected, all triploids were males. Subsequently, germ cells transplantation revealed that triploids are suitable surrogate hosts. Production of donor-derived sperm was achieved in 23% and 16% of triploids transplanted by testicular and ovarian cells, respectively. Success of the transplantation was confirmed by positive GFP signal detected in gonads of dissected fish and stripped sperm. Germline transmission was confirmed by fertilization tests followed by PCR analysis of embryos. Reproductive success of germline chimera triploids evaluated as fertilization rate and progeny development was comparable to control groups.

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