Vitrification and rapid-freezing of cumulus cells from rabbits and pigs

To use adult somatic cloning technology in animal breeding, this technology should be complemented with nuclear donor cell cryopreservation. Two different conventional nonequilibrium methods (vitrification, V: 3.58M EG and 2.82M DMSO in PBS plus 20% FCS and rapid-freezing, RF: 0.25M sucrose, 2.25M EG and 2.25M DMSO in PBS plus 20% FCS) were assayed here on different cumuli types from rabbits and pigs. In rabbits, the cell proliferation capability of fully disaggregated cumuli was not affected by cryopreservation procedures (V: 100% and RF: 82%). Vitrified samples from partially or non-disaggregated cumuli showed the lowest proliferation frequencies (4% and 0%, respectively). In pigs, differences in cell proliferation capability were only observed between vitrified non-disagreggated cumuli and vitrified or rapid-frozen, fully disaggregated cumuli (72% vs 100% or 100%, respectively; P < 0.05). In both species, in vitro cultured sub-confluent samples were able to survive to a second cryopreservation treatment, maintaining the cell proliferation capability in nearly 50% of thawed samples. In conclusion, before cryopreservation, disaggregation of cumulus cells from both species into small clusters of cells improved their viability after thawing. These results allow us to efficiently, easily and rapidly store rabbit and pig cumulus cells, from selected high-merit females.