A microplate incubation method for assessing egg quality of the barfin flounder: effects of well size and rearing medium on larval viability
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In microplate incubation, fish eggs and larvae are individually reared in small wells. This technique is useful for assessing hatchability, larval survival, and larval abnormality. Nevertheless, the optimal well size (water volume) and rearing media required to ensure full larval viability vary among species. In this study, we examined the effects of well size and rearing medium on hatchability and larval viability of the barfin flounder Verasper moseri and attempted to optimize the incubation conditions for this species. Fertilized eggs were individually stocked in 24-, 48-, and 96-well plates filled with 2, 1, and 0.25 mL seawater containing antibiotics (penicillin G potassium and streptomycin sulfate) and bovine serum albumin (BSA), both of which are required in the rearing media for other species. The eggs were incubated without replacing the medium. Hatch timing and rate were similar in all plate types. Completion of yolk resorption was delayed only in the 96-well plates. In the 24- and 48-well plates, the larvae survived more than 2 weeks after complete yolk resorption until they were starved to death. When the antibiotics were removed from the media, larval mortality increased. In contrast, removal of BSA from the media did not increase yolk sac stage larval mortality for barfin flounder unlike that reported for other species. These results indicate that the use of 24- and 48-well microplates filled with seawater containing antibiotics is optimal for barfin flounder larval viability beyond the completion of yolk resorption.
KeywordsAntibiotics Bovine serum albumin Deformity Hatch Larva Microtiter plate Survival Verasper moseri
We are grateful to Dr. Takaaki Kayaba at the Hokkaido Research Organization for helpful discussion on our study and critical reading of this manuscript. We also thank Suzuko Tanaka, Yuko Toya, and Yasuko Fuchigami at the Hokkaido National Fisheries Research Institute for technical assistance throughout the study.
This study was funded, in part, by the Ministry of Agriculture, Forestry and Fisheries, Japan (Research and Development Projects for Application in the New Policy of Agriculture, Forestry and Fisheries).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All applicable international, national, and institutional guidelines for the care and use of animals were followed.
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