Effects of Multi-Well Plate Incubation on Embryo-Larval Development in Fish: A Case Study Using Fathead Minnow (Pimephales promelas)

Rearing early-life stages of fish in multi-well (tissue culture) plates is a common practice in toxicology, developmental biology and fisheries research. However, the effects that such conditions may have on the speed of embryo-larval development is rarely examined. Here we compared rates of embryonic development in embryos of fathead minnow, a frequently-used model species in ecotoxicology, reared under standard conditions with a range of possible methods. We showed that fathead minnow embryos incubated in multi-well plates flexed less frequently as embryos, hatched smaller, later and with larger yolk-sacs, and initiated feeding later than embryos reared in large volumes with or without supplemental aeration. Developmental differences persisted in larvae reared to 20 days post-fertilization when monitoring ceased, but growth differences did not magnify and in some measurements partially resolved. To our knowledge we are the first to report effects of incubation in multi-well plates in any fish taxa. As our data revealed that the yolk-dependent stage for fathead minnow may be prolonged when reared in multi-well plates, use of plates may also allow the use of longer experiments using embryos without conflicting with existing animal welfare legislation in many countries.