Preserving the vanishing biodiversity of Indian donkeys via fibroblast cell cryobanking

Abstract

India possesses four officially registered indigenous donkey breeds, namely Halari, Kachchhi, Spiti, and Ladakhi, which represent unique genetic resources adapted to diverse agro-climatic conditions. The steep decline in donkey populations due to mechanization and changing socio-economic conditions necessitates the development of complementary ex situ conservation strategies. The present study aimed to establish and cryopreserve skin fibroblast cell lines from ear pinnae tissues of all four registered Indian donkey breeds. Ear tissue biopsies of at least 2 male and 2 female animals of each breed were collected from their representative breeding tracts. Primary cell cultures were established within 10-14 days. Cultures were expanded, characterized morphologically, and cryopreserved in liquid nitrogen using dimethyl sulfoxide-based freezing medium. Population doubling time of cells selected for freezing varied within a narrow range of 22.4-25.1 hours across all the 4 breeds. Successful preservation of viable fibroblast cultures was evident from the post-thaw viability of (>90%) after 6 months and >85% after a year. The established cell lines exhibited typical spindle-shaped morphology, rapid attachment following thawing, and sustained proliferative capacity under in vitro conditions. The generated fibroblast repository represents a secure backup of the complete diploid genome of all registered Indian donkey breeds and provides a valuable resource for future applications including genomic characterization, induced pluripotent stem cell generation, somatic cell nuclear transfer, and breed restoration programmes. This study also demonstrates the utility of somatic cell banking as a strategic component of national animal genetic resource conservation programmes.