World Aquaculture Magazine - June 2020

WWW.WA S.ORG • WORLD AQUACULTURE • JUNE 2020 57 successful in many aquaculture species (Piferrer et al. 2009, Fig. 12). In sablefish, we first developed and validated three methods for ploidy determination in whole embryos, larvae or blood samples. Next, methods were successfully developed for triploidy induction using hydrostatic pressure or cold shock. Optimized pressure and cold shock treatments induced triploidy in a high percentage of male and female fish with minimal adverse effects on survival. Diploid (control) and putative triploid sablefish were reared until reaching a size (~200 mm in length) at which gonadal development could be assessed by histology and ploidy and genotypic sex determined from blood samples. Diploid females had ovaries with well-developed primary oocytes, while triploid females had ovaries that exhibited suppressed development, with mostly empty ovarian lamellae and reduced numbers of smaller primary oocytes. Diploid and triploid males had testes that appeared similar and composed of type-A spermatogonia. One- year, post-weaning, female triploids had an average gonadosomatic index (GSI) that was reduced ~10-fold relative to that of diploid females, but there was no difference in GSI between triploid and diploid males. Triploid fish had significantly higher rates of morphological deformities and lower survival during a one-year post-stocking evaluation. Triploidy is disruptive to female gonadal development in sablefish and could be a valid approach for reproductive sterilization. However, additional work is needed to reduce deformities and evaluate the effects of triploidy on growth rate and survival in this species. The third approach to sterilization of sablefish we investigated involved a newer technique using a gene silencing (morpholino oligomer, MO) protocol (Wong and Zohar 2015b) that targets the germ cell-specific gene, dead end ( dnd ). Treatments were applied to sablefish eggs, either before or after fertilization (Fig. 13), where they were immersed in a solution containing a sablefish dnd -MO- Vivo conjugate. Eggs were incubated, hatched and reared using standard hatchery protocols until gonadal development could be assessed by histology. After gonadal differentiation, ~10 percent of fish in the pre- fertilization treatment group had gonads devoid of germ cells, whereas ~15 percent of fish had significantly reduced germ cell counts and 75 percent had apparently normal gonads. The post-fertilization treatment was less successful, however. It is possible to produce germ cell-free (completely sterile) sablefish using the dnd -MO-Vivo bath immersion technique. Ongoing work is focused on optimizing this method to generate higher proportions of germ cell-free fish and to assess the performance of these fish in aquaculture. Transgenesis, Gene Editing and Xenogenesis for Controlling Reproduction Rex Dunham, Baofeng Su, Ian Butts, Cuiyu Lu and Jeremy Gurbatow Reproduction, both excess and sparse, can limit application of aquaculture and biotechnology. Technologies are needed to allow total reproductive control of aquatic organisms for aquaculture and fisheries management. Xenogenesis is a tool being developed with several unique applications to enhance reproduction. Xenogenesis, a method of reproduction in which successive generations differ from each other, results in xenogens, organisms comprised of elements typically foreign to its species. Potential applications include resurrection of extinct species or lines of fish, rapid expansion of endangered species or populations, expansion of difficult-to-breed fish populations, as a hatchery tool to improve fry production of difficult-to-spawn fish or fish with long generation intervals, as a tool to greatly accelerate genetic/reproductive research by reducing generation intervals, clone gonads of valuable individuals and as a tool to avoid inbreeding. Our interest is to use xenogenesis as a novel tool to produce channel catfish female × blue catfish male hybrid embryos. A xenogenic catfish (triploid channel catfish male producing blue catfish sperm was mated with a normal channel catfish female, resulting in 100 percent channel catfish female × blue catfish male hybrids, but in very low numbers (Perera et al. 2017; Fig. 14). This was accomplished with fresh stem cells, but xenogenic channel catfish were also produced by injecting cryopreserved blue catfish stem cells into blastulae (Shang et al. 2015). Potential for greater fry production is possible as mating two xenogenic white catfish Ameiurus catus resulted in large numbers of channel catfish fingerlings. Identifying the appropriate time for stem cell introduction may be the key to consistently producing highly fertile xenogenic broodstock. Colonization of blue catfish stem cells in LEFT: FIGURE 12. Adam Luckenbach resects the pituitary gland of a sablefish sampled from a sterilization trial at the Northwest Fisheries Science Center, Manchester Research Station. Photo: Edward Hayman. RIGHT: FIGURE 13. Ten-Tsao Wong collects eggs stripped by Ken Massee from female sablefish broodstock at the Northwest Fisheries Science Center, Manchester Research Station. The eggs were used for gene silencing/bath immersion experiments for sterility induction. Sablefish broodstock are handled only under red light to reduce stress and avoid disruption of their reproductive cycle. Photo: Edward Hayman. ( C O N T I N U E D O N P A G E 5 8 )