Using calcein and oxytetracycline to apply a dual mark on American shad otoliths

Restoration of American shad is a high priority for fisheries agencies in the northeast and mid-Atlantic regions of the U.S.  Stocking efforts involve releasing non-feeding fry which receive immersion baths in oxytetracycline (OTC) to produce a series of otolith marks, each specific to a stocking location.  In the Susquehanna and Delaware River shad programs, as many as 14 different otolith patterns are needed to evaluate stocking efforts.  As a demonstration project, larval shad that received an OTC mark at 3 d post-hatch were obtained from the PA Fish & Boat Commission and exposed to calcein immersion at 7 d post-hatch.  Marked larvae were reared on natural plankton in a 0.25-acre pond for 55 days.  Otolith analysis on survivors showed that the majority had discernable OTC/calcein banding patterns.  Marks were more distinguishable when digitally photographed than when viewed microscopically with the naked eye.  Relative cost of the calcein treatment and wastewater disposal are factors which currently limit calcein use for shad marking.  With refinements, this technique could be used to reduce hatchery residence time for larval shad while still maintaining the required number of unique otolith banding patterns resulting in lower hatchery costs and overall greater hatchery survival of larvae.