A Baseline-Calibrated Developmental Index to Compare Incubation Protocols in a Sensitive Transgenic Zebrafish Line

Embryo incubation is often treated as a routine husbandry step in zebrafish facilities, yet sensitive transgenic lines may not respond to standard protocols like robust wild-type stocks. The effects of feasible changes in incubation medium and embryo density on developmental performance and between-plate variability were evaluated in Tg(kop:7a-dsRed-nos3′UTR), a line showing variable output under routine conditions. Embryos were incubated in E3 medium or recirculating aquaculture system (RAS) water at 25, 50, or 100 embryos per Petri dish. Survival, hatching, larval length, and morphological abnormalities were integrated into a Baseline-calibrated Developmental Index (BDI), using wild-type AB embryos as an in-house reference. All Tg(kop) conditions remained below the wild-type baseline, with mean BDI_abs values of 0.540–0.650 compared with 0.916 in wild type. E3 tended to support higher mean developmental performance, but with greater between-plate variability. RAS water produced lower mean BDI values with lower observed between-plate variability. Embryo density showed no clear directional effect under daily-care conditions. These results indicate that sensitive transgenic lines may require dedicated incubation phenotyping, and that baseline-calibrated multiparametric assessment can help distinguish protocols that differ in mean output and observed plate-to-plate variability.