Integrated phenotypic, cytogenetic, and functional characterization of skin fibroblast cultures from the Greater Caribbean manatee (Trichechus manatus manatus, Linnaeus 1758).

The Greater Caribbean manatee, recently reclassified as endangered, is a keystone species in coastal and freshwater ecosystems. Its long-term conservation requires integrative strategies that combine in situ and ex situ approaches. Although skin-derived cell cultures have been reported for other sirenian species, none have yet been established and characterized for the Greater Caribbean manatee. Thus, the aim of this study was to establish and characterize skin-derived fibroblast cultures from the Greater Caribbean manatee. Skin biopsies obtained from seven animals were used to establish primary cultures. The cells were expanded in vitro, identified as fibroblasts by vimentin immunostaining, and characterized by ultrastructural features, karyotype stability, and the absence of microbial contamination under antibiotic-free culture conditions. Fibroblasts at passages 2-10 were characterized for morphology, viability, metabolic activity, population doubling time, oxidative stress levels, mitochondrial membrane potential, and apoptotic profile. Explant-derived cultures adhered within 24 h, reached sub-confluence after a mean of 8.4 days, and a full confluence by 17.9 days, sustaining active proliferation for up to 84.6 days across ten passages. Cells displayed typical fibroblast-like morphology, high viability (≥ 84%), and stable metabolic activity and proliferation rates (P > 0.05) throughout the culture period. Reactive oxygen species levels increased significantly at passages 4, 8, and 10 relative to passage 2 (P < 0.05), accompanied by a mild reduction in mitochondrial membrane potential, while overall cellular functionality was preserved. Apoptotic and necrotic events exhibited only transient fluctuations without evidence of progressive cellular damage. Cytogenetic analyses revealed a stable karyotype of 2n = 48, with normal XX/XY constitution and no detectable chromosomal abnormalities. Cultures remained free of microbial contamination for 30 days under antibiotic-free conditions. Therefore, this study establishes the first established fibroblast culture from the Greater Caribbean manatee, offering a novel and reliable platform for functional, genomic, cytogenetic, and toxicological studies in conservation biotechnology.