A NEW PROTOCOL FOR EXPLORING THE POTENTIAL GROWTH RATE OF ZEBRAFISH LARVAE AND JUVENILES

Over the past twenty years, zebrafish have become a widely popular model organism. However, husbandry techniques, particularly in the early life stages, require further development and are lacking standardization. We describe a novel larval culture protocol that results in high survival and rapid growth during the larval and juvenile stages. Larval fish were reared in static, 3ppt saline water with a 24L:0D photoperiod from 5-21 days post-fertilization (dpf). Turbidity was maintained at 10-20 NTU with Nannochloropsis algae paste. For the first five days of feeding (5-10dpf) marine rotifers Brachionus plicatilis were maintained in the fish tank at a concentration of 200 rotifers/mL. At 11dpf, density of fish was decreased by 1/3. Fish were then fed Artemia nauplii, which were maintained at a concentration of 10/mL until 21dpf. The effect of stocking density on growth during this period was investigated. Twenty-five progenies from individual breeding pairs were stocked at various densities and reared using this method. A density of approximately 16.7 fish/L (25 fish/L from 5-10dpf) was determined to be the highest density achievable in this stagnant water system without compromising growth (Fig 1). Higher densities decreased growth but not survival.

At 21dpf, 22 progenies were transferred to a freshwater recirculating system with a 13L:11D photoperiod and stocked at a density of 20 fish/L. Fish were fed Artemia nauplii supplemented with dry feed (Otohime B1/B2). At 42dpf, fish were measured (Table 1) and density was decreased to 1.6 fish/L. Once all fish were clearly identifiable as male or female, three pairs from three progenies (total of 9 pairs) were spawned (Table 2). Sex ratio was determined in 16 progenies and the average percent male was 58±24%. These results demonstrate that the described protocol for larval rearing results in high survival and rapid growth without any negative impacts on fertility or sex differentiation.