The use of species-specific TaqMan probes for identifying early stage gadoid eggs following formaldehyde fixation

Surveys of fish eggs are increasingly being used to monitor the spawning areas and stock status of commercially important species such as Atlantic cod (Gadus morhua), but early stage cod eggs are visually indistinguishable from those of several other common co-occurring species, including haddock (Melanogrammus aeglefinus) and whiting (Merlangius merlangus). In recent surveys in the Irish and North Seas, a molecular identification technique (TaqMan multiplex real-time polymerase chain-reaction) assay has been used to overcome this problem. The method needs high-quality DNA, so the current protocol requires that individual "cod-like" eggs are "presorted" from plankton hauls on board ship and immediately preserved in ethanol. This increases seagoing staff costs, can be a difficult process at sea, and means that plankton sampling cannot be undertaken from non-specialized vessels such as fishing boats. Successful application of TaqMan probes to DNA from eggs preserved in formalin would overcome these problems, but previous attempts have resulted in poor success. In this study, batches of hatchery-sourced cod, haddock, and whiting eggs were fixed in 4% buffered formalin for up to 3 weeks, then transferred to a formaldehyde-free solution for 1, 2, or 3 months. After these periods they were assessed visually for fixation quality and analysed using species-specific TaqMan probes. Eggs, which had been fixed for up to 3 weeks in formalin, were identified successfully, although the positive rate (84-96%) was slightly lower than samples preserved throughout in ethanol (92-99%). There was no increase in the percentage of eggs misidentified comparing formalin-fixed and ethanol-preserved material. These results suggest that TaqMan probes can be applied successfully to fish eggs fixed in 4% buffered formalin for up to 3 weeks.