The current methods for cryopreservation of swine embryos are time consuming and troublesome. One of the most successful methods of cryopreservation of swine embryos is to centrifuge the cells to polarize the lipids and then use a micromanipulator to remove the lipids from the embryo (Nagashima et al., 1995;Nagashima et al., 1994). Interestingly, removal of the lipids not only permits the embryos to survive low temperatures (Nagashima et al., 1999), but in some cases they actually develop to the blastocyst stage at a higher rate (Lai et al., 2006;Li et al., 2006). Unfortunately with current procedures this results in a break in the zona pellucida. The International Embryo Transfer Society guidelines state that the zona pellucida cannot be compromised if the health status is to be maintained (Stringfellow, 1998;Wrathall and Sutmoller, 1998). This is especially important for embryos that are to be frozen and transported to another facility, as swine are generally raised in specific pathogen environments and the movement of pathogens can have a devastating effect on the recipient herd. With this background we have formulated the following thesis. Thesis: Lipids can be efficiently removed from swine embryos without damaging the zona pellucida. Such embryos can survive the rigors of cryopreservation. This leads to a single Specific Aim, with multiple sub-Aims.
Specific Aim. Develop a non-invasive method for relatively high-throughput cryopreservation of swine embryos.
Our aim i s to develop a non-invasive """"""""relatively"""""""" high-throughput method for lipid removal prior to cryopreservation. Our first sub-aim is to confirm that the amount of space in the zona pellucida relative to the zygote cytoplasm can be increased so that complete separation of the lipids and the zygote cytoplasm can be achieved by centrifugation. We propose to determine how sensitive the in vivo-produced embryo is to various osmolality conditions for a short duration. The second sub-aim will be to use the added space within the zona pellucida, caused by high osmolality treatment, to aid in completely separating the lipids within the zona pellucida (via centrifugation) prior to cryopreservation. Viability after warming will be determined. Finally, as an extension of these in vitro studies embryo transfers will be performed to assess the true viability of these cryopreserved embryos.
(provided by applicant): The results from these studies will help to improve the resources that are needed to understand human health by making it easier to work with swine as models of human disease.
| Spate, Lee D; Murphy, Clifton N; Prather, Randall S (2013) High-throughput cryopreservation of in vivo-derived swine embryos. PLoS One 8:e65545 |
| Men, H; Walters, E M; Nagashima, H et al. (2012) Emerging applications of sperm, embryo and somatic cell cryopreservation in maintenance, relocation and rederivation of swine genetics. Theriogenology 78:1720-9 |
