Evaluation of Incubation Parameters in a TUNEL assay for Detecting Apoptotic cells in Cumulus Oocyte Complexes and in vitro produced early Embryos of Buffalo

Terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) is used widely for detecting the apoptotic status in biological cells of diverse origin. Reproducing the TUNEL procedure with specialized cells like cumulus oocyte complexes (COCs) poses technical challenge in view of mammalian oocytes which undergo very dynamic transitions in the course of oocyte maturation within a short time. The current work describes a standardized TUNEL protocol where the process of fixation and permeabilization were optimized using different concentrations of paraformaldehyde and Triton X-100 with sodium citrate, respectively. Effectiveness of the procedure was validated at different time intervals of oocyte culture using known oocyte stimulators. The procedure was further validated in the in vitro produced early embryos. Consistent and specific signal could be obtained using the procedure depicting the apoptotic status of cumulus cells and early embryos at different cell stages in buffalo.