Approaches for reproductive sterilization are needed for marine aquaculture to increase sustainability and mitigate public concern associated with escapement of farmed fish and potential harm to wild stocks. One such approach is triploidization, a technique that has been widely used in freshwater fish species but not as well explored in marine species. Triploidy can be achieved by applying a "shock" to eggs shortly after fertilization to disrupt completion of meiosis and induce retention of the second polar body, which contains a complete set of chromosomes normally extruded from the early embryo. This study developed methods for triploidy induction and ploidy determination in a new marine aquaculture species, sablefish (or black cod, Anoplopoma fimbria).
Flow cytometry methods were assessed for ploidy determination in sablefish embryos, larvae, and erythrocytes. Microscopy techniques for ploidy determination, including nucleolar organizing region (NOR) analyses in larvae and erythrocyte major axis measurements in blood smears were also tested as lower cost alternatives. Experiments were conducted to establish initial methods for induction of triploidy using either hydrostatic pressure or cold shock. For each experiment, a minimum of four unique breeding crosses were made. Each consisted of ~100 ml of green eggs fertilized with 1 ml of sperm (diluted 1:30 with Cortland's solution) that were split equally into 50 ml vessels to either undergo a shock or no shock (control). The first experiment evaluated the optimal time after fertilization for application of pressure shock (10, 20, 30, or 40 min post-fertilization; mpf), the second evaluated the intensity of pressure shock (7,000, 9,000, or 11,000 psi), and the third evaluated the duration of cold shock (60, 120, or 180 min) at -1.5 ºC. Three criteria were used to evaluate the triploidy protocols relative to controls: embryo cell symmetry (proxy for embryo quality), percent survival to hatch, and triploidy rate.
Flow cytometry was effective for ploidy determination in embryos, larvae, and juvenile blood samples, NOR analysis was effective for larvae, and erythrocyte measurements were effective for juvenile blood samples. The timing of pressure shock (Exp. 1) had the greatest effect on the triploid criteria assessed, as all treatments, except 10 mpf, induced significantly lower cell symmetry and hatch rates compared to controls. Higher levels of pressure (9,000 and 11,000 psi; Exp. 2) significantly reduced hatch rates, whereas hatch and symmetry rates at 7,000 psi were not different than those of controls. Duration of cold shock (Exp. 3) had no effect on symmetry rates, however the hatch rate at 180 min was significantly lower than that of controls. For all treatments tested, 100% triploids were obtained based on NOR analyses (n=12 larvae/treatment/replicate). These results indicate that both pressure and cold shock can be used to induce triploidy in sablefish and that shock protocols applied at 10 mpf with 7,000 psi for 5 min or -1.5 ºC for 60-120 min maximize embryo quality and survival, while maintaining high levels of triploidy.