Intra-ovarian busulfan administration: an effective and relatively safe method for depleting endogenous germ cells in the female marine teleost yellow drum (Nibea albiflora).

Production of germ cell-depleted fish is of great significance for aquaculture as well as germ cell transplantation (GCT). In this study, we established an effective and safe method to deplete endogenous germ cell in adult female yellow drum (Nibea albiflora) via intra-ovarian (IO) injection of busulfan. All-female individuals were treated with busulfan (40 mg/kg body weight) through the ovarian duct at 7-day intervals for four times. No mortality was observed throughout the experimental period. Histological and VASA immunofluorescence analyses confirmed successful depletion of endogenous oocytes, while a population of VASA-positive undifferentiated germ cells was retained within the ovigerous lamellae. Quantitative real-time PCR revealed a significant downregulation of germ cell-associated genes, whereas the expression of ovarian development-associated genes was sustained. Physiological assays, including key serum and hepatic enzyme activity analyses, and intestinal microbiota profiling, showed no significant differences between busulfan-treated and control fish. These findings demonstrate that IO busulfan injection is an effective and non-lethal approach for sterilizing adult female, providing a valuable model for GCT applications and advancing reproductive manipulation techniques in marine fish.