Production of piglets after cryopreservation of embryos using a centrifugation-based method for delipation without...

It is still difficult to successfully cryopreserve in vitro-produced (IVP) swine embryos, as they are sensitive to chilling due to the abundance of intracellular lipids. Mechanical delipation through micromanipulation is successful, but this method increases the potential of pathogen transmission because of the damage inflicted upon the zona pellucida during micromanipulation, and it is labor intensive. Reported here is a method to remove the lipid of IVP porcine embryos, without significantly compromising the zona pellucida, by trypsin treating the embryos or exposing the embryo to a high-osmolality solution to enlarge the perivitelline space so that the lipid could be polarized and separated completely after subsequent centrifugation without micromanipulation. The procedures work both for nuclear transfer-derived embryos and in vitro-fertilized embryos. Both methods provide a high-throughput process that leaves the zona pellucida intact (or relatively intact for the trypsin treatment) to aid in preventing disease transmission. It is also demonstrated that this procedure results in viable piglets, a claim that could not be made in many previous reports. Although the efficiencies of cryopreservation have not been dramatically improved, these procedures allow a single person to process very large numbers of embryos without the necessity of manipulating each individual embryo on a micromanipulator. Such high-throughput processing overcomes the lack of high efficiency (i.e., the system can be overloaded with embryos for transfer to surrogates).