Bovine nuclear transfer using parthenogenetically activated oocyte as recipient cytoplast

Abstract

Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol, Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 PCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EPS) for 30 sec and then to 5 M ethylene glycol in EFS for 25 to 30 sec at 37°C. Oocytes were loaded into straws in ~2 μL of cryoprotectant and plunged directly into LN2. Warming straws and dilution of cryoprotectant was at 37°C in TCM-199 + 10% FCS + 0.25 M sucrose for 1 min and then TCM-199 + 10% FCS + 0.15 M sucrose for 5 min. Non-vitrified oocytes undergoing the same maturation protocol for both species were used as controls. Oocytes were stained with orcein for nuclear maturation and live/dead status was determined using Hoechst 33342.