Cryopreservation of Skin Fibroblast Cells from Kendrapada Sheep and Lipopolysaccharide-induced Gene Expression Studies

A study was conducted to investigate the in vitro cryopreservation and temporal gene expression variations in lipopolysaccharide (LPS)-stimulated skin fibroblasts of Kendrapada sheep. Four skin samples from two Kendrapada rams (~3 yr old) were cultured, fibroblasts were characterized morphologically, and cryopreserved in liquid nitrogen. After LPS stimulation of passage-4 of cells, gene expression analysis was carried out. Real time PCR expression of genes viz., toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (MyD88), TIR domain containing adapter inducing interferon β (TRIF), cluster of differentiation 14 (CD14), tumor necrosis factor alpha (TNFα), interferon regulatory factor 3 (IRF 3), interleukin (IL) 1, 2, 4, 8, 10 and interferon gamma (IFNγ) was carried out at 12, 24 and 36 h post stimulation (PS). Transcripts expression analysis revealed dynamic regulation over time, with an initial inflammatory surge followed by varied expression patterns. There was an increase in IL8, IRF3, and IL10 at 12 h followed by a reduction at 24 h and a mixed response at 36 h, indicating a complex regulatory landscape. The exceptionally high expression (39.26±0.29) of IL8 throughout signifies its pivotal role in the immune response, while the observed downregulation of IL10 and IL2 at 24 h hints at regulatory feedback mechanisms or adaptive processes. These findings offer crucial insights into Kendrapada sheep's immune response mechanisms, informing conservation efforts and enriching our understanding of their adaptation to diverse agro-climatic conditions.